Chimeric antigens for eliciting an immune response

ABSTRACT

Disclosed herein are compositions and methods for eliciting immune responses against antigens. In particular embodiments, the compounds and methods elicit immune responses against antigens that are otherwise recognized by the host as “self” antigens. The immune response is enhanced by presenting the host immune system with a chimeric antigen comprising an immune response domain and a target binding domain, wherein the target binding domain comprises a xenotypic antibody fragment. By virtue of the target binding domain, antigen presenting cells take up, process, and present the chimeric antigen, eliciting both a humoral and cellular immune response.

RELATED APPLICATIONS

The present invention is a continuation-in-part of co-pending U.S. Ser.No. 10/365,620, filed Feb. 13, 2003, which application claims benefit ofU.S. Provisional Application Nos. 60/390,564 filed Jun. 20, 2002 and60/423,578 filed Nov. 5, 2002. The present application is also acontinuation-in-part of co-pending international applicationPCT/IB04/000373, filed Feb. 14, 2004, which application designates theUnited States. The entire disclosure of each of these priorityapplications is hereby incorporated by reference.

I. INTRODUCTION

1. Technical Field

The present invention relates to chimeric antigens (fusion proteins) fortargeting and activating antigen presenting cells. In particular, theinvention describes compositions and methods that contain or use one ormore fusion proteins that contain a pre-selected HBV antigen or HCVantigen, and a xenotypic immunoglobulin fragment, wherein the fusionmolecule is capable of binding and activating antigen presenting cells,especially dendritic cells.

2. Background

Viral infectious diseases are major public healthcare issues. HumanHepatitis B virus (HBV) is a member of a family of DNA viruses thatprimarily infect the liver (Gust, et al., Intervirology 25:14-29 (1986).Other members of this family are woodchuck hepatitis B virus (WHV)(Summers, et al., Proc Natl Acad Sci USA 75(9): 4533-7 (1978)), duckhepatitis B virus (DHBV) (Mason, et al., J Virol 36(3): 829-36 (1980))and heron hepatitis B virus (HHBV) (Sprengel, et al., J Virol 62(10):3832-9 (1988)). These viruses share a common morphology and replicationmechanisms, but are species specific for infectivity (Marion, Prog MedVirol. 35:43-75 (1988)).

HBV primarily infects liver cells and can cause acute and chronic liverdisease resulting in cirrhosis and hepatocellular carcinoma. Infectionoccurs through blood and other body fluids. Approximately 90% of theindividuals infected by HBV are able to clear the infection, while theremaining 10% become chronic carriers of the virus with a highprobability of developing cirrhosis of the liver and hepatocellularcarcinoma. The World Health Organization statistics show that more than2 billion people have been infected by HBV and among these, 370 millionare chronically infected by the virus (Beasley, Cancer 61(10):1942-56(1988); Kane Vaccine 12:547-49 (1995)). Prophylactic vaccines based onHBV surface antigen (HBsAg) have been very effective in providingprotective immunity against HBV infections. These vaccines have beendeveloped from HBsAg purified from plasma of chronic HBV carriers,produced by recombinant DNA techniques as well as through the use ofsynthetic peptides (see, e.g. U.S. Pat. Nos. 4,599,230 and 4,599,231).These vaccines are highly effective in the prevention of infection, butare ineffective in eradicating established chronic infections.

Human Hepatitis B Virus (HBV) belongs to the family of Hepadnaviruses.Other members of this family are Duck Hepatitis B Virus (DHBV),Woodchuck Hepatitis Virus (WHV) Ground Squirrel Hepatitis B Virus (GSHV)and Heron Hepatitis B Virus (HHBV). Although these viruses have similarmorphology and replication mechanism, they are fairly species specific.Consequently, they infect only very closely related species. Theseviruses have a DNA genome ranging in size from 3.0-3.2 Kb, withoverlapping reading frames to encode several proteins. HBV genomeencodes several proteins. Among these, the surface antigens: Large(S1/S2/S), Medium (S2/S) and Small (S) are proposed to be involved inthe binding of the virus to the cellular receptors for uptake. The Coreprotein (Core) forms capsids, which encapsulate the partially doublestranded DNA genome. Polymerase/Reverse Transcriptase (Pol) protein is amultifunctional enzyme necessary for the replication of the virus. The Xprotein has been proposed to have many properties, including theactivation of Src kinases (Ganem, Science 294(5550):2299-300 (2001)).The present invention describes DNA sequences and amino acidcompositions of the surface antigen proteins S1/S2, S1/S2/S as well asCore protein fusion proteins with a xenotypic monoclonal antibody (mAb)fragment.

DHBV, another member of the hepadnaviral family, infects pekin ducks, isspecies specific, and has served as an animal model for studying thehepatitis B viruses. DHBV has a DNA genome and it codes for surfaceantigens PreS and PreS/S, Core protein (Core) and Polymerase/ReverseTranscriptase. The present invention also describes DNA sequences anddeduced amino acid sequences of fusion proteins of the PreS, PreS/S andCore proteins with a fragment of a xenotypic mAb. These fusion proteinscan be used to elicit a broad immune response in chronic viralinfections, thus as therapeutic vaccine.

Hepatitis C virus (HCV) is a member of the flaviviridae family of RNAviruses. The route of infection is via blood and body fluids and over50% of the patients become chronic carriers of the virus. Persistentinfection results in chronic active hepatitis, which may lead to livercirrhosis and hepatocellular carcinoma (Saito et. al., PNAS USA87:6547-6549 (1990)).

Approximately 170 million people worldwide are chronic carriers of HCV(Wild and Hall, Mutation Res. 462: 381-393 (2000)). There is noprophylactic vaccine available at present. Current therapy is interferonα-2b and ribavirin, either alone or as combination therapy. Thesignificant side effects for interferon treatment and the development ofmutant strains are major drawbacks to the current therapy. Moreover,interferon therapy is effective only in 20% of the patients. Therapeuticvaccines to enhance host immune system to eliminate chronic HCVinfection will be a major advancement in the treatment of this disease.

HCV genome is a positive sense single stranded RNA molecule ofapproximately 9.5 Kb in length. This RNA, which contains both 5′ and 3′untranslated regions, codes for a single polyprotein that is cleavedinto individual proteins and catalyzed by both viral and host proteases(Clarke, J. Gen. Virol. 78: 2397-2410 (1997)). The structural proteinsare Core, Envelope E1 & E2 and P7. The non-structural proteins are NS2,NS3, NS4A, NS4B, NS5A and NS5B. Core forms capsids. E1, E2 are envelopeproteins, also called “Hypervariable region” due to the high rate ofmutations. NS3 is a Serine Protease, the target of several proteaseinhibitors as antivirals for HCV. NS5B is the RNA Polymerase enzyme.NS5A has recently been suggested to have a direct role in thereplication of the virus in the host by counteracting the interferonresponse (Tan, and Katze, Virology 284:1-12 (2001)) that augments theimmune function.

When a healthy host (human or animal) encounters an antigen (such as aprotein derived from a bacterium, virus or parasite), normally the hostinitiates an immune response. This immune response can be a humoralresponse and/or a cellular response. In the humoral response antibodiesare produced by B cells and are secreted into the blood and/or lymph inresponse to an antigenic stimulus. The antibody then neutralizes theantigen, e.g. a virus, by binding specifically to antigens on itssurface, marking it for destruction by phagocytotic cells and/orcomplement-mediated mechanisms. The cellular response is characterizedby the selection and expansion of specific helper and cytotoxicT-lymphocytes capable of directly eliminating the cells that contain theantigen.

In many individuals, the immune system does not respond to certainantigens. When an antigen does not stimulate the production of aspecific antibody and/or killer T cells, the immune system is unable toprevent the resultant disease. As a result, the infectious agent, e.g. avirus, can establish a chronic infection and the host immune systembecomes tolerant to the antigens produced by the virus. The mechanism bywhich the virus evades the host immune machinery is not clearlyestablished. The best-known examples of chronic viral infections includeHepatitis B, Hepatitis C, Human Immunodeficiency Virus, Human PapillomaVirus and Herpes Simplex Virus.

In chronic states of viral infections, the virus escapes the host immunesystem. Viral antigens are recognized as “self,” and thus not recognizedby the antigen-presenting cells. The lack of proper presentation of theappropriate viral antigen to the host immune system may be acontributing factor. The success in eliminating the virus will resultfrom the manner in which the antigen is processed and presented by theantigen presenting cells (APCs) and the involvement of the regulatoryand cytotoxic T cells. The major participant in this process is theDendritic Cell (DC), which captures and processes antigens, expresseslymphocyte co-stimulatory molecules, migrates to lymphoid organs, andsecretes cytokines to initiate immune responses. Dendritic cells alsocontrol the proliferation of B and T lymphocytes, which are themediators of immunity (Steinman, et al., Hum Immunol 60(7):562-7(1999)). The generation of a cytotoxic T lymphocyte (CTL) response iscritical in the elimination of the virus infected cells and thus a cureof the infection.

Antigen presenting cells process the encountered antigens differentlydepending on the localization of the antigen (Steinman et al., 1999,supra). Exogenous antigens are processed within the endosomes of the APCand the generated peptide fragments are presented on the surface of thecell complexed with Major Histocompatibility Complex (MHC) Class II. Thepresentation of this complex to CD4⁺ T cells stimulates the CD4⁺ Thelper cells. As a result, cytokines secreted by the helper cellsstimulate B cells to produce antibodies against the exogenous antigen(humoral response). Immunizations using antigens typically generateantibody response through this endosomal antigen processing pathway.

On the other hand, intracellular antigens are processed in theproteasome and the resulting peptide fragments are presented ascomplexes with MHC Class I on the surface of APCs. Following binding ofthis complex to the co-receptor CD8 molecule, antigen presentation toCD8⁺ T cells occurs which results in a cytotoxic T lymphocyte (CTL)immune response to remove the host cells that carry the antigen.

In patients with chronic viral infections, since the virus is activelyreplicating, viral antigens will be produced within the host cell.Secreted antigens will be present in the circulation. As an example, inthe case of chronic HBV carriers, virions and HBV surface antigens and asurrogate for core antigens (in the form of e-antigen) can be detectedin the blood. An effective therapeutic vaccine should be able to inducestrong CTL responses against an intracellular antigen or an antigendelivered into the appropriate cellular compartment so as to activatethe MHC Class I processing pathway. An effective prophylactic vaccinewill induce a strong humoral immune response, thus producing antibodiesto neutralize circulating virions.

These findings would suggest that a therapeutic vaccine that can inducea strong CTL response should be processed through the proteasomalpathway and presented via the MHC Class I (Larsson, et al., TrendsImmunol 22(3):141-8 (2001)). This can be achieved either by producingthe antigen within the host cell, or it can be delivered to theappropriate cellular compartment so that it gets processed and presentedso as to elicit a cellular response. Several approaches have beendocumented in the literature for the intracellular delivery of theantigen. Among these, viral vectors (Lorenz, et al., Hum Gene Ther10(7):1095-103 (1999)), the use of cDNA-transfected cells (Donnelly, etal., Annu Rev Immunol 15: 617-48 (1997)) as well as the expression ofthe antigen through injected cDNA vectors (Lai and Bennett, Crit RevImmunol 18(5): 449-84 (1998); U.S. Pat. No. 5,589,466), have beendocumented. Further, DNA vaccines expressing antigens targeted todendritic cells have been described (You, et al., Cancer Res61:3704-3711 (2001)).

Delivery vehicles capable of carrying the antigens to the cytosoliccompartment of the cell for MHC Class I pathway processing have alsobeen used. The use of adjuvants to achieve the same goal has beendescribed in detail by (Hilgers, et al., Vaccine 17(3):219-28 (1999))Another approach is the use of biodegradable microspheres in thecytoplasmic delivery of antigens (Newman, et al., J Biomed Mater Res50(4):591-7 (2000)), exemplified by the generation of a Th1 immuneresponse against ovalbumin peptide (Newman, et al., J Control Release54(1):49-59 (1998); Newman, et al., J Biomed Mater Res 50(4): 591-7(2000)). It has also been shown that PLGA nanospheres are taken up bythe most potent antigen presenting cells, dendritic cells (Newman, etal., J Biomed Mater Res 60(3): 480-6 (2002)).

Dendritic cells derived from blood monocytes, by virtue of theircapability as professional antigen presenting cells have been shown tohave great potential as immune modulators that stimulate primary T cellresponse (Steinman, et al., Hum Immunol 60(7): 562-7 (1999); Banchereauand Steinman, Nature 392(6673):245-52 (1998)). This property of the DCsto capture, process, present the antigen and stimulate naive T cells hasmade them very important tools for therapeutic vaccine development(Laupeze, et al., Hum Immunol 60(7): 591-7 (1999)). Targeting of theantigen to the DCs is the crucial step in the antigen presentation andthe presence of receptors on the DCs for the Fc region of monoclonalantibodies have been exploited for this purpose (Regnault, et al., J ExpMed 189(2): 371-80 (1999)). Examples of this approach include ovariancancer mAb-B43.13, Anti-PSA antibody as well as Anti-HBV antibodyantigen complexes (Wen, et al., Int Rev Immunol 18(3): 251-8 (1999)).Cancer immunotherapy using DCs loaded with tumor associated antigenshave been shown to produce tumor-specific immune responses andanti-tumor activity (Campton, et al., J Invest Dermatol 115(1):57-61(2000); Fong and Engleman, Annu Rev Immunol 18: 245-73 (2000)).Promising results were obtained in clinical trials in vivo usingtumor-antigen-pulsed DCs (Tarte and Klein, Leukemia 13(5): 653-63(1999)). These studies clearly demonstrate the efficacy of using DCs togenerate immune responses against cancer antigens.

The primary goal in antiviral therapy is the complete elimination of theinfectious virus. In the case of chronic hepatitis B, this will resultin the eradication of hepatitis B viremia, the arrest of progressiveliver injury, normalization of liver transaminase activity, resolutionof hepatic inflammation, elimination of HBV cccDNA (covalently closedcircular DNA) reservoir, and improve the quality of life of the patient.

Two forms of antiviral therapies are currently in use for the treatmentof chronic hepatitis B infections. First, antiviral compounds,particularly nucleoside analogues, which are DNA chain terminators,suppress the viral replication resulting in a decrease in HBV DNA andHBV antigens. The effectiveness of the antiviral compound depends on thelevel of immune help from the host. The second therapy involves the useof immune modulators, such as interferons (e.g., interferon α-2b), tostimulate the immune system into mounting a generalized humoral andcellular response against the viral infection.

The most widely used antiviral nucleoside agent is lamivudine, acytosine analogue that acts as a chain terminator and inhibits HBVreplication. The drug is well tolerated and has marked virus-suppressiveactivity in the majority of patients; complete clearance of the virusoccurs if the patient has elevated levels of liver inflammatory enzymes.This suggests that a strong involvement of the host immune system isneeded to clear the HBV infection. While lamivudine suppresses HBVreplication in HBV carriers, replication recurs if therapy is stopped.The emergence of resistant mutants also is a possibility.

Interferons are biologic response modifiers that have a variety oftherapeutic activities, including antiviral, immunomodulatory, andantiproliferative effects. They enhance T cell helper activity, causematuration of B lymphocytes, inhibit T cell suppressors, and enhance HLAtype I expression. While interferons have only mild to moderatevirus-suppressive activities, they induce a generalized, non-specificbut clinically important immune response in receptive individuals.

The indications for interferon therapy are specific: patients must beHbeAg-positive, have detectable HBV DNA in the serum, and have a serumALT level double the upper limit of normal. When these patients aretreated with a standard course of interferon-α therapy (30-35 MUinterferon/week for 16-26 weeks), the response rate is 40-50%. Aresponse is defined as loss of HBeAg, development of anti-HBe, loss ofHBV DNA (by non-PCR assays), and normalization of ALT. A sustainedresponse encompasses the foregoing outcomes plus generating effectiveimmune responses. The responses are usually durable, are associated withimprovement in liver histology, and produce a better long-term outcome,e.g., with fewer patients progressing to cirrhosis and/or hepatocellularcarcinoma.

For most chronic hepatitis B patients, monotherapy with a standard 16week course of interferon-α or a one year course of lamivudine iseffective in only 30-40% of patients. It is reasonable to assume thatcombining the antiviral effect of one drug with a second agent promotingimmune modulation may improve the response rate beyond that seen witheither agent alone. However, in order to produce more effective andspecific immune responses against chronic hepatitis B infections thanare currently achievable with current biological response modifiers, anagent that induces highly specific cellular immune responses directedagainst cells harboring the viruses, viral antigens or cccDNA must beemployed. The chimeric antigens described in the present invention aresuch agents.

The goals of treatment of chronic hepatitis C include eradication of theHCV infection, improvement or normalization of liver histology,prevention of progression of the viral liver infection to cirrhosis andhepatocellular carcinoma, extension of patient survival, improvement ofthe quality of life, and a reduction in the size of the infectious poolof hepatitis C virus patients in order to reduce the wide spreadtransmission of the disease.

Two forms of treatment of chronic hepatitis C are currently in use:pegylated interferon-α used alone and conventional or pegylatedinterferon-α used in combination with ribavirin. Ribavirin is a purinenucleoside analogue that as monotherapy has little effect on HCVviremia, despite the fact that it significantly reduces serum ALT levelsin some patients. While the exact nature of the synergism of ribavirinand interferon has not been elucidated, the efficacy of the combinationexceeds that of either agent used alone.

While the mechanism of action of ribavirin in hepatitis C infection isnot understood, a number of mechanisms have been proposed including: (a)the enhancement of host T cell-mediated immunity against viral infectionthrough switching the T cell phenotype from type 2 to type 1; (b) theinhibition of the host enzyme inosine monophosphate dehydrogenase(IMPDH); (c) the direct inhibition of HCV, including NS5B-encodedRNA-dependent RNA polymerase (RdRp); and (d) its action as an RNAmutagen that drives a rapidly mutating RNA virus over the threshold to“error catastrophe.”

Interferons are biologic response modifiers that have a variety oftherapeutic activities including antiviral, immunomodulatory, andantiproliferative effects. They enhance T cell helper activity, causematuration of B lymphocytes, inhibit T cell suppressors, and enhance HLAtype I expression. While interferons have only mild to moderatevirus-suppressive activities, they induce a generalized, non-specificbut clinically important immune response in receptive individuals thatreduces viral levels.

The treatment options for previously untreated patients with hepatitis Cinclude pegylated interferon monotherapy and a combination ofconventional or pegylated interferon with ribavirin. The overallsustained response rate (SR) of ribavirin combined with conventionalinterferon α-2b therapy for 48 weeks is about 40%. The SR for patientsinfected with genotype 2 or 3 patients is about 60%, whereas the SR isabout 30% for patients infected with genotype 1 (Lauer and Walker, NEJMed 345:41-42 (2001)). However, the combination is associated withsignificantly more side effects than conventional interferon alone. Up20% of patients receiving the combination required a reduction of doseor discontinuation of therapy because of the side effects. Nevertheless,the combination represents a significant improvement in the treatment ofchronic hepatitis C and has become the current standard of care.

Conventional interferon-α is rapidly cleared from the circulation by thekidneys. During the first 12 hours after interferon administration,interferon-α causes the viral levels to decrease significantly, butafter that time, the viral levels begin to increase because of low bloodlevels of interferon. Sustained viral suppression can be achieved by theadministration of pegylated interferon, which is administered only oncea week and produces constant blood levels of interferon for 7 days.Thus, there is no need for the daily dosing that is required withconventional interferon. Per the Peg-Intron® product insert, the overallSR in previously untreated chronic hepatitis C patients who receivedpegylated interferon for 48 weeks was about 39%, which is comparable tothe previously reported SR with combined conventional interferon α-2band ribavirin combination (Rebetron®).

In studies comparing combined pegylated interferon and ribavirin to theRebetron® combination, the pegylated interferon and ribavirincombinations appeared to be more effective, especially in patientsinfected with HCV genotype 1. For patients infected with this genotype,the sustained response rate (SR) was about 45% for the pegylatedinterferon and ribavirin combination compared with about 35% for theRebetron® combination. As expected, the overall response rates in HCVgenotype 2 or 3 patients for each of these treatment groups were betterthan those obtained with HCV genotype 1 patients (SR 60% to 80%).

In a trial comparing Rebetron® with varying doses of PEG-Intron®(pegylated interferon α-2a) and ribavirin, the patients werepredominately male Caucasians, more frequently infected with HCVgenotype 1, and had a mean age of 44 years (Mann, et al., Lancet358:958-965 (2001)). The best-sustained virologic response of 54% wasobtained with PEG-Intron® plus ribavirin given for 48 weeks. Patientswith HCV genotype 1 had an SR of about 40%, while patients with HCVgenotypes 2 and 3 after 48 weeks of therapy had the best sustainedvirologic response rate of approximately 80%, regardless of whether theyreceived Rebetron® or PEG-Intron® and ribavirin. Adverse events in thePEG-Intron® plus ribavirin group that were more than 10% more frequentthan in the standard interferon and ribavirin group included fever,nausea, and injection site reaction. Twelve percent of patients onPEG-Intron® plus ribavirin required dose modifications due to an adverseevent, while 34% had dose modifications due to a lab abnormality.

In another large, multinational, multicenter trial of plus ribavirin,the three arms of the study were Pegasys® plus placebo, standardinterferon α-2b plus ribavirin (Rebetron®), and Pegasys® plus ribavirin,which were all given for 48 weeks (Fried et al., N Engl J. Med.347(13):975-982 (2002)). There were 1,149 predominantly male patients inthe trial with an average age of about 40; 12% to 15% of patients hadcirrhosis and approximately two-thirds had infection with HCVgenotype 1. The overall sustained virologic response with Pegasys® plusribavirin was 56% compared to 30% in the Pegasys® plus placebo group,and 45% in the standard interferon α-2b plus ribavirin (Rebetron®)group. Patients with HCV genotype I had a 46% SR with Pegasys® plusribavirin, while patients with genotypes 2 and 3 had a 76% SR. Fever,myalgia, rigors, and depression were relatively less frequent withPegasys® plus ribavirin compared to standard interferon α-2b plusribavirin (Rebetron®). In the Pegasys® plus ribavirin group, the rate ofdiscontinuation of therapy due to an adverse event was 7% and due to alab abnormality was 3%.

Despite vigorous treatment with the current standard combination therapyof interferon-α and ribavirin, there are still a large proportion ofpatients with chronic HCV who do not respond. In order to produce animproved sustained response rate in the treatment of chronic hepatitis Cinfection, an agent that induces highly specific cellular immuneresponses directed against cells harboring the hepatitis C viruses mustbe employed. Such an agent is the chimeric antigen hepatitis C vaccine.

There is no prophylactic vaccine available to prevent new HCVinfections. The attempts to develop preventative vaccines using theenvelope proteins of HCV have been unsuccessful due to the high rate ofmutation of the virus. Similarly, no therapeutic vaccine is availablefor the treatment of existing and/or chronic HCV infections. Chimericantigens described in the present invention incorporating immunologicalattributes of HBV antigen and xenotypic monoclonal antibody have beenshown to elicit both a strong humoral and strong cellular immuneresponse against viral antigen in animal models. Chimeric antigensdescribed in the present invention incorporating HCV antigens andxenotypic monoclonal antibody fragment could be used for prophylaxisand/or treatment.

II. SUMMARY OF THE INVENTION

The present invention pertains to compositions and methods for targetingand activating antigen presenting cells, one of the first steps ineliciting an immune response. The compositions of the present inventioninclude a novel class of bifunctional molecules (hereinafter designatedas “chimeric antigens”) that include an immune response domain (IRD),for example a recombinant protein, linked to a target binding domain(TBD), for example, a xenotypic antibody fragment portion. Morespecifically, the chimeric antigens are molecules that couple viralantigens, such as Hepatitis B Core or surface proteins, to a xenotypicFc fragment, such as a murine immunoglobulin G fragment.

The compositions and methods of the present invention are useful fortargeting and activating antigen presenting cells. The present inventionmay be useful for inducing cellular and humoral host immune responsesagainst any viral antigen associated with a chronic viral infection,including but not limited to Hepatitis B, Hepatitis C, HumanImmunodeficiency Virus, Human Papilloma Virus (HPV), and Herpes SimplexVirus. The invention may also be applicable to prophylactic vaccines,especially for viral disease, and to all autologous antigens in diseasessuch as cancer and autoimmune disorders.

The present invention relates to chronic infectious diseases, and inparticular to chronic HBV infections. The presentation of HBV antigensto elicit a cellular or humoral immune response by the use of vaccinemolecules designed to target the vaccines to DCs whereby theHBV-associated antigens treated as “self” during the chronic infectionwill be recognized as “foreign” and the host's immune system will mounta CTL response to eliminate HBV-infected cells is provided. At the sametime, the antibody response to the circulating HBV antigen will bind tothe antigen and remove it from the circulation. Accordingly, the presentinvention is designed to produce vaccines that can induce a broad immuneresponse in patients who have chronic viral infections such as HBV.

One or more embodiments of the present invention include one or morechimeric antigens suitable for initiating an immune response againstHepatitis B virus (HBV). In these embodiments of the invention, selectedHBV antigens are linked to fragments of xenotypic antibodies. Theresulting chimeric antigens are capable of targeting and activatingantigen presenting cells, such as dendritic cells.

One or more embodiments of the present invention include one or morechimeric antigens suitable for initiating an immune response againstHepatitis C virus (HCV). In these embodiments of the invention, selectedHCV antigens are linked to fragments of xenotypic antibodies. Theresulting chimeric antigens are capable of targeting and activatingantigen presenting cells, such as dendritic cells.

The present invention also includes methods for cloning and producingfusion proteins in a heterologous expression system. In preferredembodiments of the invention, the cloning and production methodsintroduce unique post-translational modifications including, but notlimited to glycosylation on the expressed fusion proteins.

In order to provide efficient presentation of the antigens, theinventors have developed a novel murine monoclonal antibody Fcfragment-antigen (viral antigenic protein/peptide) fusion protein. Thismolecule, by virtue of the Fc fragment is recognized at a higherefficiency by the antigen-presenting cells (dendritic cells) asxenotypic, and the viral antigen is processed and presented as complexeswith Major Histocompatibility Complex (MHC) Class I. This processing andantigen presentation is expected to result in the up-regulation of theresponse by cytotoxic T-lymphocytes, resulting in the elimination ofvirus-infected cell population. In addition, due to antigen presentationby MHC Class II molecules, humoral response also aids in the antibodyresponse to the viral infection.

The bifunctional nature of the molecule helps to target the antigen tothe proper antigen-presenting cells (dendritic cells), making it aunique approach in the therapy of chronic infectious diseases byspecifically targeting the antigen presenting cells with the mosteffective stoichiometry of antigen to antibody. This is useful to thedevelopment of therapeutic vaccines to cure chronic viral infectionssuch as Hepatitis B, Hepatitis C, Human Immunodeficiency Virus, HumanPapilloma Virus and Herpes Simplex Virus, and may also be applicable toall autologous antigens in diseases such as cancer and autoimmunedisorders.

The administration of these fusion proteins can elicit a broad immuneresponse from the host, including both cellular and humoral responses.Thus, they can be used as therapeutic vaccines to treat subjects thatare immune tolerant to a particular infection.

One aspect of the invention provides chimeric antigens for eliciting animmune response, said chimeric antigen comprising an immune responsedomain and a target binding domain, wherein the target binding domaincomprises a xenotypic antibody fragment. The immune response can be ahumoral and/or cellular response, elicited in vivo or ex vivo. In thecase where a cellular response is elicited, the immune response can be aTh1 response, a Th2 response and/or a CTL response. The chimeric antigencan comprise more than one immune response domain, or an immune responsedomain that can confer immunity to more than one antigen. In certainembodiments, the chimeric antigen of the invention further comprises a6×His-peptide, a protease cleavage site, and/or a linker for linking theimmune response domain and the target binding domain. In preferredembodiments, the immune response domain comprises one or moreimmunogenic portions of a protein selected from the group consisting ofa hepatitis B virus (HBV) protein, a duck hepatitis B virus (DHBV)protein, a hepatitis C virus (HCV) protein or a protein from HumanPapilloma Virus (HPV), Human Immunodeficiency Virus (HIV), HerpesSimplex Virus (HSV) or a cancer antigen. In other preferred embodiments,the xenotypic antibody fragment comprises an Fc fragment, an antibodyhinge region, a portion of or an entire C_(H)1 domain, a portion of oran entire C_(H)2 domain and/or a portion of or an entire C_(H)3 domain.In a particularly preferred embodiment, the xenotypic antibody fragmentis a mouse antibody fragment. The target binding domain, optionally, canalso comprise a 6×His tag, a protease cleavage site (preferably a rTEVprotease cleavage site) and/or a linker for linking the immune responsedomain and the target binding domain. The linker may be leucine zippers,biotin/avidin or a covalent peptide linkage, such as SRPQGGGS (SEQ IDNO: 28). In a preferred embodiment, the chimeric antigen isglycosylated. The immune response domain and/or the target bindingdomain can be glycosylated. In a particularly preferred embodiment, thechimeric antigen is mannose glycosylated by either high mannoseglycosylation or by pauci mannose glycosylation.

Another aspect of the invention provides chimeric antigens for elicitingan immune response to HBV, said chimeric antigen comprising an immuneresponse domain and a target binding domain, wherein the immune responsedomain comprises a protein selected from the group consisting of a HBVCore protein, a HBV S protein, a HBV S1 protein, a HBV S2 protein, andcombinations thereof, and wherein the target binding domain comprises axenotypic antibody fragment. The immune response can be a humoral and/orcellular response, elicited in vivo or ex vivo. When a cellular responseis elicited, the immune response can be a Th1 response and or a Th2response.

Yet another aspect of the invention relates to chimeric antigens foreliciting an immune response to DHBV, said chimeric antigens comprisingan immune response domain and a target binding domain, wherein theimmune response domain comprises a protein selected from the groupconsisting of a DHBV Core protein, a DHBV Pre-S protein, a DHBV PreS/Sprotein, and combinations thereof, and wherein the target binding domaincomprises a xenotypic antibody fragment. The immune response can be ahumoral and/or cellular response, elicited in vivo or ex vivo. When acellular response is elicited, the immune response can be a Th1 responseand or a Th2 response.

An aspect of the invention provides chimeric antigens for eliciting animmune response to HCV, said chimeric antigens comprising an immuneresponse domain and a target binding domain, wherein the immune responsedomain comprises a protein selected from the group consisting of a HCVCore (1-191) protein, a HCV Core (1-177) protein, a HCV E1 protein, aHCV E2 protein, a HCV E1-E2 protein, a HCV NS3 protein, a HCV NS5Aprotein, and combinations thereof, and wherein the target binding domaincomprises a xenotypic antibody fragment. The immune response can be ahumoral and/or cellular response, elicited in vivo or ex vivo. When acellular response is elicited, the immune response can be a Th1 responseand or a Th2 response.

Another aspect of the invention provides methods of enhancing antigenpresentation in antigen presenting cells, said method comprisingadministering, to the antigen presenting cells, a chimeric antigen thatcomprises an immune response domain and a target binding domain, whereinthe target binding domain comprises a xenotypic antibody fragment. In apreferred embodiment, the antigen presenting cells are dendritic cells.

An aspect of the invention relates to methods of activating antigenpresenting cells comprising contacting an antigen presenting cell with achimeric antigen that comprises an immune response domain and a targetbinding domain, wherein the target binding domain comprises a xenotypicantibody fragment. The antigen presenting cell can be contacted with thechimeric antigen in vivo or ex vivo. In another preferred embodiment,the contacting takes place in a human.

Yet another aspect of the invention provides methods of eliciting animmune response, said method comprising administering to a subject acomposition comprising a chimeric antigen that comprises an immuneresponse domain and a target binding domain, wherein the target bindingdomain comprises a xenotypic antibody fragment. The immune response canbe a humoral and/or cellular response, elicited in vivo or ex vivo. Whena cellular response is elicited, the immune response can be a Th1response and/or a Th2 response. In a preferred embodiment, the cellularimmune response is a Th1 response, a Th2 response or both a Th1 and aTh2 response.

Another aspect of the invention provides methods of treatingimmune-treatable conditions comprising administering, to a subject inneed thereof, a chimeric antigen that comprises an immune responsedomain and a target binding domain, wherein the target binding domaincomprises a xenotypic antibody fragment. Preferably, theimmune-treatable condition is an infection or a cancer. More preferably,the immune-treatable condition is a viral infection, even morepreferably, a chronic viral infection. Most preferably, theimmune-treatable condition is a chronic hepatitis B viral infection or achronic hepatitis C viral infection. For the treatment of HBV,preferably the immune response domain comprises an antigenic portion ofa protein selected from the group consisting of a HBV Core protein, aHBV S protein, a HBV S1 protein, a HBV S2 protein, and combinationsthereof. For the treatment of HCV, preferably the immune response domaincomprises an antigenic portion of a protein selected from the groupconsisting of a HCV Core (1-191) protein, a HCV Core (1-177) protein, aHCV E1 protein, a HCV E2 protein, a HCV E1-E2 protein, a HCV NS3protein, a HCV NS5A protein, and combinations thereof.

Another aspect of the invention provides methods of vaccinating asubject against an infection comprising administering to the subject achimeric antigen that comprises an immune response domain and a targetbinding domain, wherein the target binding domain comprises a xenotypicantibody fragment. Preferably, the infection is a viral infection. Themethod of the invention can prophylactically vaccinate the animalagainst the infection or therapeutically vaccinate a subject having apreexisting infection.

Yet another aspect of the invention provides polynucleotides encoding achimeric antigen, said polynucleotide comprising a first polynucleotideportion encoding an immune response domain and a second polynucleotideportion encoding a target binding domain, wherein the target bindingdomain comprises a xenotypic antibody fragment. In one embodiment, thepolynucleotide comprises a nucleotide sequence selected from the groupconsisting of nucleotides 1 to 1326 of SEQ ID NO: 31, nucleotides 1 to2004 of SEQ ID NO: 35, nucleotides 1 to 1350 of SEQ ID NO: 39,nucleotides 1 to 1293 of SEQ ID NO: 43, nucleotides 1 to 1794 of SEQ IDNO: 47, nucleotides 1 to 1581 of SEQ ID NO: 51, nucleotides 1 to 1389 ofSEQ ID NO: 57, nucleotides 1 to 1347 of SEQ ID NO: 61, nucleotides 1 to2157 of SEQ ID NO: 65, nucleotides 1 to 1395 of SEQ ID NO: 69,nucleotides 1 to 1905 of SEQ ID NO: 73 and nucleotides 1 to 2484 of SEQID NO: 77. Yet another embodiment provides polynucleotides that encodesa chimeric antigen that is at least 90% identical to an amino acidsequence selected from the group consisting of amino acids 1 to 442 ofSEQ ID NO: 32, amino acids 1 to 668 of SEQ ID NO: 36, amino acids 1 to450 of SEQ ID NO: 40, amino acids 1 to 431 of SEQ ID NO: 44, amino acids1 to 598 of SEQ ID NO: 48, amino acids 1 to 527 of SEQ ID NO: 52, aminoacids 1 to 463 of SEQ ID NO: 58, amino acids 1 to 449 of SEQ ID NO: 62,amino acids 1 to 719 of SEQ ID NO: 66, amino acids 1 to 465 of SEQ IDNO: 70, amino acids 1 to 635 of SEQ ID NO: 74 and amino acids 1 to 828of SEQ ID NO: 78. One preferred embodiment includes polynucleotides thatselectively hybridize under stringent conditions to a polynucleotidehaving a nucleotide sequence selected from the group consisting of SEQID NO: 31, 35, 39, 43, 47, 51, 57, 61, 65, 69, 73 and 77.

The invention also provides microorganisms and cell lines comprising apolynucleotide of the invention. Preferably, the microorganism or cellline is a eukaryotic microorganism or cell line. More preferably themicroorganism or cell line is a non-mammalian eukaryotic microorganismor cell line. In a preferred embodiment the microorganism or cell lineis a yeast, a plant cell line or an insect cell line. In a particularlypreferred embodiment, the cell line is an insect cell line selected fromthe group consisting of Sf9, Sf21, Drosophila S2 and High Five™.

One aspect of the invention provides methods for producing a chimericantigen comprising (a) providing a microorganism or cell line thatcomprises a polynucleotide encoding a chimeric antigen; and (b)culturing said microorganism or cell line under conditions whereby thechimeric antigen is expressed. Preferably, the microorganism or cellline is eukaryotic, more preferably a non-mammalian eukaryotic,microorganism or cell line. In a preferred embodiment, the microorganismor cell line is a yeast, a plant cell line or an insect cell line. In aparticularly preferred embodiment, the cell line is an insect cell lineselected from the group consisting of Sf9, Sf21, Drosophila S2 and HighFive™. In another particularly preferred embodiment, the yeast isselected from the group consisting of Saccharomyces cerevisiae,Schizosaccharomyces pombe, Pichia pastoris, and Pichia august.

Another aspect of the invention relates to articles of manufacturecomprising a chimeric antigen of the invention and instructions foradministering the chimeric antigen to a subject, in need thereof.

Yet another aspect of the invention relates to a pharmaceuticalcomposition comprising a pharmaceutically acceptable excipient and achimeric antigen that comprises an immune response domain and a targetbinding domain comprising a xenotypic antibody fragment. Preferably thepharmaceutical composition is formulated for parenteral, transdermal,intradermal, nasal, pulmonary or oral administration.

III. DESCRIPTION OF DRAWINGS

FIG. 1 a provides a schematic diagram illustrating the structure of thechimeric antigen of the present invention as a monomer, wherein thechimeric antigen has two portions, namely an antigen and a xenotypicmurine Fc fragment. In a preferred embodiment, a hinge region ispresent. FIG. 1 b provides a schematic diagram illustrating thestructure of the chimeric antigen of FIG. 1 in its normal, assembledstate as a dimer. FIG. 1 b illustrates a particularly preferredembodiment, in which the chimeric antigen comprises a 6×His tag andpeptide linker in addition to the IRD and TBD.

FIG. 2 a provides a schematic diagram illustrating the structure of anexemplary modified chimeric antigen as a monomer, wherein the chimericantigen has two portions, namely a modified viral antigen portion whichincorporates any viral antigen or antigens, antigenic protein fragmentsor peptides, or any of these with glycosylation at specific sites, and axenotypic binding agent, namely a murine Fc fragment with the hingeregion present. FIG. 2 b is a schematic diagram illustrating thestructure of the modified chimeric antigen of FIG. 2 a in its normal,assembled state as a dimer. The abbreviations “Ag1,” “Ag2,” and “Ag3”represent different viral antigenic peptides or proteins.

FIG. 3 a provides a schematic diagram illustrating the structure of amodified biotinylated immune response domain comprising an antigen and afusion protein of a streptavidin and a target binding domain comprisinga Fc fragment with the hinge region present. FIG. 3 b provides aschematic diagram illustrating the structure of the modified chimericantigen of FIG. 3 in its normal, assembled state as a dimer.

FIG. 4 is a schematic diagram illustrating a recombinant bacmid, capableof expressing a chimeric antigen.

FIG. 5 is a schematic embodiment of TBD of the present invention.

FIG. 6 a provides the nucleotide sequences of the open reading frameencoding the TBD of FIG. 5 (SEQ ID NO: 29). FIG. 6 b provides the aminoacid sequence of the TBD of FIG. 5 (SEQ ID NO: 30).

FIG. 7 provides a schematic embodiment of an exemplary chimeric antigenof the present invention, suitable for use with an insect cellexpression system.

FIG. 8 a provides the nucleotide and deduced amino acid sequences of thechimeric antigen molecule of FIG. 7 (SEQ ID NO: 31). FIG. 8 b providesthe amino acid sequence of the chimeric antigen of FIG. 7 (SEQ ID NO:32).

FIG. 9 shows the nucleotide (FIG. 9 a; SEQ ID NO: 33) and deduced aminoacid (FIG. 9 b; SEQ ID NO: 34) sequences of HBV S1/S2 protein, expressedas described in Example 2.

FIG. 10 provides a schematic embodiment of an exemplary chimeric antigenof the present invention, illustrating an exemplary IRD of the presentinvention.

FIG. 11 shows the nucleotide (FIG. 11 a; SEQ ID NO: 35) and deducedamino acid (FIG. 11 b; SEQ ID NO: 36) sequences of the chimeric antigenmolecule of FIG. 10.

FIG. 12 shows the nucleotide (FIG. 12 a; SEQ ID NO: 37) and deducedamino acid (FIG. 12 b; SEQ ID NO: 38) sequences of the HBV S1/S2/Sprotein, expressed as described in Example 3.

FIG. 13 is a schematic embodiment of an exemplary chimeric antigen ofthe present invention, illustrating an exemplary IRD of the presentinvention.

FIG. 14 shows the nucleotide (FIG. 14 a; SEQ ID NO: 39) and deducedamino acid (FIG. 14 b; SEQ ID NO: 40) sequences of the chimeric antigenmolecule of FIG. 13.

FIG. 15 shows the nucleotide (FIG. 15 a; SEQ ID NO: 41) and deducedamino acid (FIG. 15 b; SEQ ID NO: 42) sequences of the HBV Core protein,expressed as described in Example 4.

FIG. 16 is a schematic embodiment of an exemplary chimeric antigen ofthe present invention, illustrating an exemplary IRD of the presentinvention.

FIG. 17 shows the nucleotide (FIG. 17 a; SEQ ID NO: 43) and deducedamino acid (FIG. 17 b; SEQ ID NO: 44) sequences of the chimeric antigenmolecule of FIG. 16.

FIG. 18 shows the nucleotide (FIG. 18 a; SEQ ID NO: 45) and deducedamino acid (FIG. 18 b; SEQ ID NO: 46) sequences of the DHBV PreSprotein, expressed as described in Example 5.

FIG. 19 is a schematic embodiment of an exemplary chimeric antigen ofthe present invention, illustrating an exemplary IRD of the presentinvention.

FIG. 20 shows the nucleotide (FIG. 20 a; SEQ ID NO: 47) and deducedamino acid (FIG. 20 b; SEQ ID NO: 48) sequences of the chimeric antigenmolecule of FIG. 19.

FIG. 21 shows the nucleotide (FIG. 21 a; SEQ ID NO: 49) and deducedamino acid (FIG. 21 b; SEQ ID NO: 50) sequences of the DHBV PreS/Sprotein, expressed as described in Example 6.

FIG. 22 is a schematic embodiment of an exemplary chimeric antigen ofthe present invention, illustrating an exemplary IRD of the presentinvention.

FIG. 23 shows the nucleotide (FIG. 23 a; SEQ ID NO: 51) and deducedamino acid (FIG. 23 b; SEQ ID NO: 52) sequences of the chimeric antigenmolecule of FIG. 22.

FIG. 24 shows the nucleotide (FIG. 24 a; SEQ ID NO: 53) and deducedamino acid (FIG. 24 b; SEQ ID NO: 54) sequences of the DHBV Coreprotein, expressed as described in Example 7.

FIG. 25 shows that a chimeric antigen embodiment of the invention can betaken up by dendritic cells.

FIG. 26 shows that dendritic cells maturation is higher in the presenceof a chimeric antigen of the present invention (S1/S2-TBD), as comparedto the target binding domain (TBD) alone, or the immune response domain(S1/S2) alone.

FIG. 27 shows the expression of MHC Class II by dendritic cells inresponse to the chimeric antigen (S1/S2-TBD), the target binding domainalone (TBD) or the immune response domain alone (S1/S2).

FIG. 28 shows that a cellular response is generated after contact withdendritic cells activated with a chimeric antigen of the presentinvention.

FIG. 29 shows T cell stimulation by a chimeric antigen of the presentinvention over a period of 2-4 days.

FIG. 30 shows a time course of expression of antigen binding receptorson maturing dendritic cells.

FIG. 31 shows a time course of expression of various dendritic cellsactivation markers.

FIG. 32 shows the comparison of binding of HBV S1/S2-TBD, IgG1, and IgG2to dendritic cells over time.

FIG. 33 shows a comparison of HBV S1/S2-TBD, IgG1, and IgG2a binding tomaturing dendritic cells on day 1.

FIG. 34 shows the comparison of HBV S1/S2-TBD, IgG1, and IgG2a bindingto maturing dendritic cells on day 4.

FIG. 35 shows the comparison of uptake between HBV S1/S2-TBD, IgG1, andIgG2 as a function of concentration.

FIG. 36 shows the correlation of HBV S1/S2-TBD binding to CD32 and CD206expression on dendritic cells.

FIG. 37 demonstrates that the binding of HBV S1/S2-TBD to CD32 and CD206receptors on dendritic cells is abolished by Fcγ fragment.

FIG. 38 shows that glycosylation of S 1/S2 antigen increases the uptakeby dendritic cells via the CD206 receptor.

FIG. 39 shows an increase in intracellular interferon-γ positive T cellsafter antigen presentation.

FIG. 40 shows an increase in secretion of interferon-γ after antigenpresentation.

FIG. 41 shows an increase in intracellular interferon-γ positive T cellsas a function of S1/S2-TBD concentration

FIG. 42 shows interferon-γ secretion by T cells as a function of S1/S2-TBD concentration.

FIG. 43 shows the effect of glycosylation on intracellular interferon-γproduction in T cells.

FIG. 44 shows the effect of glycosylation on interferon-γ secretion by Tcells.

FIG. 45 shows the nucleotide (FIG. 45 a; SEQ ID NO: 55) and amino acid(FIG. 45 b; SEQ ID NO: 56) sequences of the ORF of HCV Core (1-191) inthe plasmid pFastBac HTa-HCV.

FIG. 46 shows the nucleotide (FIG. 46 a; SEQ ID NO: 57) and amino acid(FIG. 46 b; SEQ ID NO: 58) sequences of the ORF of HCV Core-TBD in theplasmid pFastBac HTa-HCV-TBD.

FIG. 47 shows the nucleotide (FIG. 47 a; SEQ ID NO: 59) and amino acid(FIG. 47 b; SEQ ID NO: 60) sequences of the ORF of HCV Core (1-177) inthe plasmid pFastBac HTa-HCV-Core (1-177).

FIG. 48 shows the nucleotide (FIG. 48 a; SEQ ID NO: 61) and amino acid(FIG. 48 b; SEQ ID NO: 62) sequences of the ORF of HCV Core-TBD proteinin the plasmid pFastBac HTa-HCV-Core-TBD.

FIG. 49 shows the nucleotide (FIG. 49 a; SEQ ID NO: 63) and amino acid(FIG. 49 b; SEQ ID NO: 64) sequences of the ORF of HCV NS5A in theplasmid pFastBac HTa-HCV-NS5A.

FIG. 50 shows the nucleotide (FIG. 50 a; SEQ ID NO: 65) and amino acid(FIG. 50 b; SEQ ID NO: 66) sequences of the ORF of HCV NS5A-TBD in theplasmid pFastBac HTa-HCV-NS5A-TBD

FIG. 51 shows the nucleotide (FIG. 51 a; SEQ ID NO: 67) and amino acid(FIG. 51 b; SEQ ID NO: 68) sequences of the ORF of HCV E1 in the plasmidpFastBac HTa-HCV-E1.

FIG. 52 shows the nucleotide (FIG. 52 a; SEQ ID NO: 69) and amino acid(FIG. 52 b; SEQ ID NO: 70) sequences of the ORF of HCV E1-TBD in theplasmid pFastBac HTa-HCV-E1-TBD.

FIG. 53 shows the nucleotide (FIG. 53 a; SEQ ID NO: 71) and amino acid(FIG. 53 b; SEQ ID NO: 72) sequences of the ORF of HCV E2 in the plasmidpFastBac HTa-HCV-E2.

FIG. 54 shows the nucleotide (FIG. 54 a; SEQ ID NO: 73) and amino acid(FIG. 54 b; SEQ ID NO: 74) sequences of the ORF of HCV E2-TBD in theplasmid pFastBac HTa-HCV-E2-TBD.

FIG. 55 shows the nucleotide (FIG. 55 a; SEQ ID NO: 75) and amino acid(FIG. 55 b; SEQ ID NO: 76) sequences of the ORF of HCV E1/E2 in theplasmid pFastBac HTa-HCV-E1/E2.

FIG. 56 shows the nucleotide (FIG. 56 a; SEQ ID NO: 77) and amino acid(FIG. 56 b; SEQ ID NO: 78) sequences of the ORF of HCV E1/E2-TBD in theplasmid pFastBac HTa-HCV-E1/E2-TBD.

IV. DETAILED DESCRIPTION A. Overview

Disclosed herein are compositions and methods for eliciting immuneresponses against antigens. In particular embodiments, the compounds andmethods elicit immune responses against antigens that are otherwiserecognized by the host as “self” antigens. The immune response isenhanced by presenting the host immune system with a chimeric antigencomprising an immune response domain and a target binding domain,wherein the target binding domain comprises a xenotypic antibodyfragment. By virtue of the target binding domain, antigen presentingcells take up, process and present the chimeric antigen, eliciting botha humoral and cellular immune response.

B. Definitions

Prior to describing the invention in further detail, the terms used inthis application are defined as follows unless otherwise indicated.

“Antibody” refers to an immunoglobulin molecule produced by B lymphoidcells with a specific amino acid sequence evoked in humans or otheranimals by an antigen (immunogen). These molecules are characterized byreacting specifically with the antigen, each being defined in terms ofthe other.

“Antibody response” or “humoral response” refers to a type of immuneresponse in which antibodies are produced by B lymphoid cells and aresecreted into the blood and/or lymph in response to an antigenicstimulus. In a properly functioning immune response, the antibody bindsspecifically to antigens on the surface of cells (e.g., a pathogen),marking the cell for destruction by phagocytotic cells and/orcomplement-mediated mechanisms. Antibodies also circulate systemicallyand can bind to free virions. This antibody binding can neutralize thevirion and prevent it from infecting a cell as well as marking thevirion for elimination from circulation by phagocytosis or filtration inthe kidneys.

“Antigen” refers to any substance that, as a result of coming in contactwith appropriate cells, induces a state of sensitivity and/or immuneresponsiveness and that reacts in a demonstrable way with antibodiesand/or immune cells of the sensitized subject in vivo or in vitro.

“Antigen-presenting cell” refers to the accessory cells ofantigen-inductive events that function primarily by handling andpresenting antigen to lymphocytes. The interaction of antigen presentingcells (APC) with antigens is an essential step in immune inductionbecause it enables lymphocytes to encounter and recognize antigenicmolecules and to become activated. Exemplary APCs include macrophages,Langerhans-dendritic cells, Follicular dendritic cells, and B cells.

“B cell” refers to a type of lymphocyte that produces immunoglobulins orantibodies that interact with antigens.

“C_(H)1 region” refers to a region of the heavy chain constant domain onthe antigen binding fragment of an antibody.

“Cellular response” or “cellular host response” refers to a type ofimmune response mediated by specific helper and killer T cells capableof directly or indirectly eliminating virally infected or cancerouscells.

As used herein, the term “chimeric antigen” refers to a polypeptidecomprising an immune response domain and a target binding domain. Theimmune response domain and target binding domains may be directly orindirectly linked by covalent or non-covalent means. “Complex” or“antigen-antibody complex” refers to the product of the reaction betweenan antibody and an antigen. Complexes formed with polyvalent antigenstend to be insoluble in aqueous systems.

“Cytotoxic T-lymphocyte” is a specialized type of lymphocyte capable ofdestroying foreign cells and host cells infected with the infectiousagents that produce viral antigens.

“Epitope” refers to the simplest form of an antigenic determinant, on acomplex antigen molecule; this is the specific portion of an antigenthat is recognized by an immunoglobulin or T cell receptor.

“Fusion protein” refers to a protein formed by expression of a hybridgene made by combining two or more gene sequences.

“Hinge region” refers to the portion of an antibody that connects theFab fragment to the Fc fragment; the hinge region contains disulfidebonds that covalently link the two heavy chains.

The term “homolog” refers to a molecule which exhibits homology toanother molecule, by for example, having sequences of chemical residuesthat are the same or similar at corresponding positions. The phrase “%homologous” or “% homology” refers to the percent of nucleotides oramino acids at the same position of homologous polynucleotides orpolypeptides that are identical or similar. For example, if 75 of 80residues in two proteins are identical, the two proteins are 93.75%homologous. Percent homology can be determined using various softwareprograms known to one of skill in the art.

“Host” refers to a warm-blooded animal, including a human, which suffersfrom an immune-treatable condition, such as an infection or a cancer. Asused herein, “host” also refers to a warm-blooded animal, including ahuman, to which a chimeric antigen is administered.

In the context of this invention, “hybridization” means the pairing ofcomplementary strands of oligomeric compounds. In the present invention,the preferred mechanism of pairing involves hydrogen bonding, which maybe Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding,between complementary nucleoside or nucleotide bases (nucleobases) ofthe strands of oligomeric compounds. For example, adenine and thymineare complementary nucleobases that pair through the formation ofhydrogen bonds. Hybridization can occur under varying circumstances. Theterms “hybridize”, “hybridizing”, “hybridizes” and the like, used in thecontext of polynucleotides, are meant to refer to conventionalhybridization conditions, preferably such as hybridization in 50%formamide/6×SSC/0.1% SDS/100 μg/ml mDNA, in which temperatures forhybridization are above 37° C. and temperatures for washing in0.1×SSC/0.1% SDS are above 55° C.

“Immunity” or “immune response” refers to the body's response to anantigen. In particular embodiments, it refers to the ability of the bodyto resist or protect itself against infectious disease.

“Immune Response Domain (IRD)” refers to the variously configuredantigenic portion of a bifunctional molecule. The IRD comprises one ormore antigens or one or more recombinant antigens. Preferred viralantigens include, but are not limited to, HBV PreS1/S2 HBV PreS1/S2/S,HBV Core, HBV Core ctm (C-terminal modified), HBV e-antigen, HBVPolymerase, HCV Core, HCV E1-E2, HCV E1, HCV E2, HCV NS3-serineprotease, HCV NS5A and NS4A, HIV gp120 and HSV Alkaline nuclease and HPVAntigens.

As used herein, the phrase “immune-treatable condition” refers to acondition or disease that can be prevented, inhibited or relieved byeliciting or modulating an immune response in the subject.

“Lymphocyte” refers to a subset of nucleated cells found in the blood,which mediate specific immune responses.

“Monoclonal antibody” or “mAb” refers to an antibody produced from aclone or genetically homogenous population of fused hybrid cells, i.e.,a hybridoma cell. Hybrid cells are cloned to establish cells linesproducing a specific monoclonal antibody that is chemically andimmunologically homogenous, i.e., that recognizes only one type ofantigen.

“Peptide linkage” or “peptide bond” refers to two or more amino acidscovalently joined by a substituted amide linkage between the α-aminogroup of one amino acid and the α-carboxyl group of another amino acid.

A “pharmaceutical excipient” comprises a material such as an adjuvant, acarrier, a pH-adjusting and buffering agent, a tonicity adjusting agent,a wetting agent, a preservative, and the like.

“Pharmaceutically acceptable” refers to a non-toxic composition that isphysiologically compatible with humans or other animals.

The term “polynucleotide” as used herein refers to a polymeric form ofnucleotides of any length, either ribonucleotides ordeoxyribonucleotides. This term refers only to the primary structure ofthe molecule. Thus, this term includes double- and single-stranded DNAand RNA. It also includes known types of modifications, for example,labels which are known in the art, methylation, “caps”, substitution ofone or more of the naturally occurring nucleotides with an analog,internucleotide modifications such as, for example, those with unchargedlinkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates,carbamates, etc.) and with charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.), those containing pendant moieties, such as,for example proteins (including e.g., nucleases, toxins, antibodies,signal peptides, poly-L-lysine, etc.), those with intercalators (e.g.,acridine, psoralen, etc.), those containing chelators (e.g., metals,radioactive metals, boron, oxidative metals, etc.), those containingalkylators, those with modified linkages (e.g., alpha anomeric nucleicacids, etc.), as well as unmodified forms of the polynucleotide.

“Protease cleavage site” refers to a site where proteolytic enzymeshydrolize (break) polypeptide chains.

In the present invention, the phrase “stringent hybridizationconditions” or “stringent conditions” refers to conditions under which acompound of the invention will hybridize to its target sequence, but toa minimal number of other sequences.

The term “subject” refers to any warm-blooded animal, preferably ahuman.

“Tag” refers to a marker or marker sequence used to isolate or purify amolecule containing the tag. An exemplary tag includes a 6×His tag.

“T cell” refers to a type of lymphocyte responsible for antigen-specificcellular interactions, and which mediates humoral and cellular immuneresponses.

“Target Binding Domain (TBD)” refers to a region of an immunoglobulinheavy chain constant region.

The phrase “therapeutically effective amount” refers to an amount ofchimeric antigen, or polynucleotide encoding a chimeric antigen,sufficient to elicit an effective B cell, cytotoxic T lymphocyte (CTL)and/or helper T lymphocyte (Th) response to the antigen and to block orto cure or at least partially arrest or slow symptoms and/orcomplications of a disease or disorder.

The terms “treating” and “treatment” as used herein cover any treatmentof a condition treatable by a chimeric antigen in an animal,particularly a human, and include: (i) preventing the condition fromoccurring in a subject which may be predisposed to the condition but hasnot yet been diagnosed as having it; (ii) inhibiting the condition,e.g., arresting or slowing its development; or (iii) relieving thecondition, e.g., causing regression of the condition or its symptoms

“Xenotypic” refers to originating from a different species other thanthe host. For example, a recombinantly expressed antibody cloned from amouse genome would be xenotypic to a human but not to a mouse,regardless of whether that recombinantly expressed antibody was producedin a bacterial, insect or mouse cell.

C. Chimeric Antigens

A composition of the present invention includes a chimeric antigencomprising an immune response domain (IRD) and a target binding domain(TBD). In preferred embodiments of the invention, the protein portion iscapable of inducing humoral and/or T cell responses, and the targetbinding portion is capable of binding an antigen presenting cell, suchas a dendritic cell. The chimeric antigen of the present invention mayalso include one or more of the following: a hinge region of animmunoglobulin, a C_(H)1 region of an immunoglobulin, a peptide linker,a protease cleavage site, and a tag suitable for use with a purificationprotocol. A chimeric antigen of the present invention is capable ofbinding to and activating an antigen presenting cell.

In some embodiments of the invention, the IRD of the chimeric antigenincludes one or more proteins selected from the group comprising: one ormore HBV proteins, one or more recombinant HBV proteins, one or more HCVproteins, or one or more recombinant HCV proteins.

In yet another embodiment of the invention, the IRD of the chimericantigen includes a 6×His-peptide fused to one or more HBV proteins, oneor more recombinant HBV proteins, one or more HCV proteins, or one ormore recombinant HCV proteins.

In preferred embodiments of the invention, the target binding domain ofthe chimeric antigen is an antibody fragment xenotypic to the host. Forexample, if the host is a human, an exemplary xenotypic antibodyfragment is a non-human animal antibody fragment, such as from a mouse.In the preferred embodiments of the invention, the xenotypic antibodyfragment comprises a murine Fc fragment. In the most preferredembodiments of the invention, the target binding domain comprises axenotypic Fc fragment, a hinge region, a C_(H)1 region, and a peptidelinkage suitable for linking the target binding domain to the IRD.

The present invention also comprises the use of linking molecules tojoin the IRD to the TBD. Exemplary linker molecules include leucinezippers, and biotin/avidin.

In one embodiment, the chimeric antigen of the present invention is afusion protein having two portions, namely an IRD containing anantigenic sequence (such as a viral antigen(s)), and a TBD containing axenotypic Fc fragment. The xenotypic murine Fc fragment with the hingeregion present recruits the antigen-presenting cells, specificallydendritic cells, to take up the chimeric antigen. The binding region ofthe chimeric antigen thus targets antigen-presenting cells specifically.The internal machinery of the APC then processes the IRD to form anactivated APC. The activated APC must then be capable of contacting andactivating immune response cells for generating humoral and cellularimmune responses to clear infected cells.

In a further embodiment, the chimeric antigen is a fusion protein havingtwo portions, namely a modified viral antigen or antigens, antigenicprotein fragments or peptides, or any of these with glycosylation atspecific sites, and a xenotypic murine Fc fragment with the hinge regionpresent, which can also be, optionally, glycosylated.

In yet another embodiment, the invention provides a further modifiedchimeric antigen, wherein the antigen is biotinylated and the Fcfragment is generated with streptavidin as a fusion protein tofacilitate the production of a wide assortment of antigen-Fc conjugates.

In yet another embodiment, the invention provides an association betweenthe antigen and the antibody Fc fragment through chemical conjugation.

An embodiment of the present invention includes the use of recombinantantigens of HBV, HCV, or DHBV fused to a xenotypic antibody fragment bymolecular biological techniques, production of the fusion proteins in abaculovirus expression system and their use as therapeutic vaccinesagainst chronic HBV and HCV infections. The present invention providesan efficient method to deliver a hitherto unrecognized antigen to APCsin vivo so as to generate a broad immune response, a Th1 responseinvolving CTLs and a Th2 (antibody) response. The immunogenicity of thepre-selected viral antigen unrecognized by the host immune system isincreased due to the presence of the xenotypic antibody fragment as wellas by the presence of specific glycosylation introduced in the insectcell expression system. The antigen-antibody fragment fusion protein,due to the presence of the antibody component, will bind to specificreceptors present on various immune cell types including dendriticcells, macrophages, B cells and granulocytes. The fusion proteinsadministered to either humans or animals will be taken up by the APCs,especially DCs, will be hydrolyzed to small peptides and presented onthe cell surface, complexed with MHC Class I and/or MHC Class II, whichcan elicit a broad immune response and clear the viral infection.

As used herein, the term “Target Binding Domain (TBD)” refers to aregion of an immunoglobulin heavy chain constant region, which is anantibody fragment capable of binding to an Fc receptor on an APC. Inaccordance with the present invention, the TBD is a protein capable ofbinding to an Fc receptor on an APC, particularly a dendritic cell, andis subsequently transported into the APC by receptor-mediated uptake. Inaccordance with the present invention, the presence of an Fc fragmentaugments the uptake of the chimeric antigen through the Fc receptor onantigen-presenting cells, specifically dendritic cells. By virtue of thespecific uptake, the viral antigen is processed and presented asforeign; thus, an immune response is effectively elicited to thepreviously tolerated viral antigen.

Also, in accordance with the present invention, the chimeric antigen,preferably, is capable of binding to a macrophage mannose receptor. Themacrophage mannose receptor (MMR), also known as CD206, is expressed onantigen presenting cells (APC) such as dendritic cells (DC). Thismolecule is a member of the C-type lectin family of endocytic receptors.Mannosylated chimeric antigen can be bound and internalized by CD206. Ingeneral, exogenous antigen is thought to be processed and presentedprimarily through the MHC class II pathway. However, in the case oftargeting through CD206, there is evidence that both the MHC class I andclass II pathways are involved (Apostolopoulos et al., Eur. J. Immunol.30:1714 (2000); Apostolopoulos and McKenzie, Curr. Mol. Med. 1:469(2001); Ramakrishna et al., J. Immunol. 172:2845-2852 (2004)). Thus,monocyte-derived dendritic cells loaded with chimeric antigen thatspecifically targeted CD206 will induce both a potent class I-dependentCD8⁺ CTL response and a class II-dependent proliferative T helperresponse (Ramakrishna et al., supra (2004)).

An exemplary TBD is derived from Mouse anti-HBVsAg mAb (Hybridoma 2C12)as cloned in pFastBac HTa expression vector, and expressed in a HighFive™ insect cell expression system (Invitrogen). This TBD consists ofpart of C_(H)1, and Hinge-C_(H)2-C_(H)3 from N-terminal to C-terminal ofthe mouse anti-HBVs Ag mAb. The constant region of the IgG1 molecule forthe practice of the present invention contains a linker peptide, part ofC_(H)1-hinge and the regions C_(H)2 and C_(H)3. The hinge region portionof the monomeric TBD can form disulphide bonds with a second TBDmolecule. FIG. 5 illustrates a schematic representation of a TBDmolecule. The protein is expressed as an N-terminal fusion protein witha 6×His tag, a seven amino acid rTEV (recombinant tobacco etch virus)protease cleavage site and the N-terminal fusion of the Target BindingDomain (TBD) of the xenotypic (murine) mAb raised against HBV sAg(Hybridoma 2C12). The exemplary TBD is a fragment of the constant chainof the IgG1 mAb from 2C12 with the sequence of amino acids comprisingthe 8 amino acid peptide linker, five amino acids of the C_(H)1 region,the hinge sequences, C_(H)2 and C_(H)3 region sequences (FIG. 5) and tenadditional amino acids from the expression vector. The exemplary TBDfragment defined herein forms the parent molecule for the generation offusion proteins with antigens derived from viruses or other infectiousagents. FIG. 1 b depicts the formation of dimeric chimeric antigenmolecule formed via intermolecular disulphide bonds. FIG. 6 shows thenucleotide sequence of the Open Reading Frame (ORF) encoding theexemplary TBD protein and the deduced amino acid sequence as defined inFIG. 5.

FIG. 7 shows a schematic representation of an exemplary chimeric antigenvaccine molecule, as produced in the insect cell expression system. Thismolecule is a fusion protein of N-terminal 6×His tag, rTEV proteasecleavage site, HBV S1/S2 antigen, linker peptide, a part of the C_(H)1as well as C_(H)2 and C_(H)3 domains of the mouse monoclonal antibodyfrom 2C12 plus eight additional amino acids introduced as a cloningartifact. Cleavage and purification will result in the generation of HBVS1/S2-TBD molecule. FIG. 8 shows the nucleotide and amino acid sequencesof the HBV S 1/S2-TBD chimeric antigen molecule. FIG. 9 shows thenucleotide and the deduced amino acid sequences of the expressed HBVS1/S2 protein.

FIG. 10 shows a schematic representation of the fusion protein of HBVS1/S2/S-TBD. This molecule is a fusion protein of N-terminal 6×His tag,rTEV protease cleavage site, HBV S1/S2/S antigen, linker peptide, a partof the C_(H)1 as well as C_(H)2 and C_(H)3 domains of the mousemonoclonal antibody from 2C12 plus eight additional amino acidsintroduced as a cloning artifact. FIG. 11 shows the nucleotide anddeduced amino acid sequences of the ORF of the fusion protein. FIG. 12shows the nucleotide and deduced amino acid sequences of the HBV S1/S2/Sprotein.

FIG. 13 illustrates the fusion protein of HBV Core-TBD molecule asexpressed in the insect cells. This molecule is a fusion protein ofN-terminal 6×His tag, rTEV protease cleavage site, HBV S1/S2 Core,linker peptide, a part of the C_(H)1 as well as C_(H)2 and C_(H)3domains of the mouse monoclonal antibody from 2C12 plus eight additionalamino acids introduced as a cloning artifact. FIG. 14 shows thenucleotide and amino acid sequences in the ORF of the fusion protein.FIG. 15 shows the nucleotide and deduced amino acid sequences of the HBVCore protein.

Another embodiment of the present invention involves the production anduse of fusion proteins generated from Duck Hepatitis B Virus (DHBV)antigens and murine TBD. DHBV has been used as a very versatile animalmodel for the development of therapies for HBV, its human counterpart.DHBV genome encodes Surface antigens (PreS/S), the Core protein (Core),which form capsids, and the polymerase enzyme, which serves multiplefunctions.

FIG. 16 depicts a schematic representation of the fusion protein of DHBVPreS-TBD, as produced in High Five™ (Invitrogen) insect cell expressionsystem. This molecule is a fusion protein of N-terminal 6×His tag, rTEVprotease cleavage site, DHBV PreS, linker peptide, a part of the C_(H)1as well as C_(H)2 and C_(H)3 domains of the mouse monoclonal antibodyfrom 2C12 plus eight additional amino acids introduced as a cloningartifact. The nucleotide and deduced amino acid sequences of the ORF ofthe fusion protein as cloned in the plasmid pFastBac HTa are shown inFIG. 17. The nucleotide and deduced amino acid sequences of the DHBVPreS protein are shown in FIG. 18.

FIG. 19 shows schematically, another embodiment of the present inventionviz. DHBV PreS/S-TBD. This molecule is a fusion protein of N-terminal6×His tag, rTEV protease cleavage site, DHBV PreS/S, linker peptide, apart of the C_(H)1 as well as C_(H)2 and C_(H)3 domains of the mousemonoclonal antibody from 2C12 plus eight additional amino acidsintroduced as a cloning artifact. The nucleotide and amino acidsequences are presented in FIG. 20. The nucleotide and deduced aminoacid sequences of PreS/S are presented in FIG. 21.

FIG. 22 shows a schematic representation of the fusion protein of DHBVCore-TBD. This molecule is a fusion protein of N-terminal 6×His tag,rTEV protease cleavage site, DHBV Core, linker peptide, a part of theC_(H)1 as well as C_(H)2 and C_(H)3 domains of the mouse monoclonalantibody from 2C12 plus eight additional amino acids introduced as acloning artifact. FIG. 23 shows the nucleotide and deduced amino acidsequences of the DHBV Core-TBD fusion protein. The nucleotide anddeduced amino acid sequences of DHBV Core protein are shown in FIG. 24.

D. Novel Polynucleotides

Another aspect of the invention provides polynucleotides encoding achimeric antigen comprising a first polynucleotide portion encoding animmune response domain and a second polynucleotide portion encoding atarget binding domain. The first and second polynucleotide portions maybe located on the same or different nucleotide chains.

The invention provides polynucleotides corresponding or complementary togenes encoding chimeric antigens, mRNAs, and/or coding sequences,preferably in isolated form, including polynucleotides encoding chimericantigen variant proteins; DNA, RNA, DNA/RNA hybrids, and relatedmolecules, polynucleotides or oligonucleotides complementary or havingat least a 90% homology to the genes encoding a chimeric antigen or mRNAsequences or parts thereof; and polynucleotides or oligonucleotides thathybridize to the genes encoding a chimeric antigen, mRNAs, or tochimeric antigen-encoding polynucleotides.

Additionally, the invention includes analogs of the genes encoding achimeric antigen specifically disclosed herein. Analogs include, e.g.,mutants, that retain the ability to elicit an immune response, andpreferably have a homology of at least 80%, more preferably 90%, andmost preferably 95% to any of polynucleotides encoding a chimericantigen, as specifically described by SEQ ID NOs: 31, 35, 39, 43, 47,51, 57, 61, 65, 69, 73 and 77. Typically, such analogs differ by only 1to 10 codon changes. Examples include polypeptides with minor amino acidvariations from the natural amino acid sequence of a viral antigen or ofan antibody fragment; in particular, conservative amino acidreplacements. Conservative replacements are those that take place withina family of amino acids that are related in their side chains.Genetically-encoded amino acids are generally divided into fourfamilies: (1) acidic=aspartate, glutamate; (2) basic=lysine, arginine,histidine; (3) non-polar=alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine, tryptophan; and (4) uncharged polar=glycine,asparagine, glutamine, cystine, serine, threonine, tyrosine.Phenylalanine, tryptophan, and tyrosine are sometimes classified jointlyas aromatic amino acids. For example, it is reasonable to expect that anisolated replacement of a leucine with an isoleucine or valine, anaspartate with a glutamate, a threonine with a serine, or a similarconservative replacement of an amino acid with a structurally relatedamino acid will not have a major effect on biological activity.Polypeptide molecules having substantially the same amino acid sequenceas any of the polypeptides disclosed in any one of SEQ ID NOs: 32, 36,40, 44, 48, 52, 58, 62, 66, 70, 74 and 78 but possessing minor aminoacid substitutions that do not substantially affect the ability of thechimeric antigens to elicit an immune response, are within thedefinition of a chimeric antigen. Derivatives include aggregativeconjugates with other chimeric antigen molecules and covalent conjugateswith unrelated chemical moieties. Covalent derivatives are prepared bylinkage of functionalities to groups that are found in chimeric antigenamino acid chains or at the N- or C-terminal residues by means known inthe art.

Amino acid abbreviations are provided in Table 1.

TABLE 1 Amino Acid Abbreviations Alanine Ala A Arginine Arg R AsparagineAsn N Aspartate Asp D Cysteine Cys C Glutamate Glu E Glutamine Gln QGlycine Gly G Histidine His H Isoleucine Ile I Leucine Leu L Lysine LysK Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser SThreonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V

Conservative amino acid substitutions can be made in a protein withoutaltering either the conformation or the function of the protein.Proteins of the invention can comprise 1 to 15 conservativesubstitutions. Such changes include substituting any of isoleucine (I),valine (V), and leucine (L) for any other of these hydrophobic aminoacids; aspartic acid (D) for glutamic acid (E) and vice versa; glutamine(Q) for asparagine (N) and vice versa; and serine (S) for threonine (T)and vice versa. Other substitutions can also be considered conservative,depending on the environment of the particular amino acid and its rolein the three dimensional structure of the protein. For example, glycine(G) and alanine (A) can frequently be interchangeable, as can alanine(A) and valine (V). Methionine (M), which is relatively hydrophobic, canfrequently be interchanged with leucine and isoleucine, and sometimeswith valine. Lysine (K) and arginine (R) are frequently interchangeablein locations in which the significant feature of the amino acid residueis its charge and the differing pK's of these two amino acid residuesare not significant Still other changes can be considered “conservative”in particular environments (see, e.g. Biochemistry 4^(th) Ed., LubertStryer ed. (W.H. Freeman and Co.), pages 18-23; Henikoff and Henikoff,Proc Nat'l Acad Sci USA 89:10915-10919 (1992); Lei et al., J Biol Chem270(20):11882-6 (1995)).

The invention also includes polynucleotides that selectively hybridizeto polynucleotides that encode chimeric antigens. Preferably apolynucleotide of the invention will hybridize under stringentconditions to a sequence selected from SEQ ID NOs: 31, 35, 39, 43, 47,51, 57, 61, 65, 69, 73 and 77. Stringency of hybridization reactions isreadily determinable by one of ordinary skill in the art and generallyis an empirical calculation dependent upon probe length, washingtemperature, and salt concentration. In general longer probes requirehigher temperatures for proper annealing, while shorter probes needlower temperatures. Hybridization generally depends on the ability ofdenatured nucleic acid sequences to re-anneal when complementary strandsare present in an environment below their melting temperature. Thehigher the degree of desired homology between the probe and hybridizablesequence, the higher the relative temperature that can be used. As aresult, it follows that higher relative temperatures would tend to makethe reaction conditions more stringent, while lower temperatures lessso. For additional details and explanation of stringency ofhybridization reactions, see, e.g., Ausubel et al., Current Protocols inMolecular Biology, Wiley Interscience Publishers, (©1995, asSupplemented April 2004, Supplement 66) at pages 2.9.1-2.10.8 and4.9.1-4.9.13.

“Stringent conditions” or “high stringency conditions”, as definedherein, are identified by, but not limited to, those that (1) employ lowionic strength and high temperature for washing, for example 0.015 Msodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at50° C.; (2) employ, during hybridization, a denaturing agent, such asformamide, for example, 50% (v/v) formamide with 0.1% bovine serumalbumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphatebuffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at42° C.; or (3) employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodiumcitrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate,5×Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS,and 10% dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC(sodium chloride/sodium citrate) and 50% formamide at 55° C., followedby a high-stringency wash consisting of 0.1×SSC containing EDTA at 55°C. “Moderately stringent conditions” are described by, but not limitedto, those in Sambrook et al., Molecular Cloning: A Laboratory Manual,2^(nd) Ed., New York: Cold Spring Harbor Press, 1989, and include theuse of washing solution and hybridization conditions (e.g., temperature,ionic strength and % SDS) less stringent than those described above. Anexample of moderately stringent conditions is overnight incubation at37° C. in a solution comprising: 20% formamide, 5×SSC (150 mM NaCl, 15mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt'ssolution, 10% dextran sulfate, and 20 mg/mL denatured sheared salmonsperm DNA, followed by washing the filters in 1×SSC at about 37-50° C.The skilled artisan will recognize how to adjust the temperature, ionicstrength, etc. as necessary to accommodate factors such as probe lengthand the like.

Embodiments of a polynucleotide of the invention include: apolynucleotide encoding a chimeric antigen having a sequence selectedfrom any of the sequences shown in FIGS. 32, 36, 40, 44, 48, 52, 58, 62,66, 70, 74 and 78, a nucleotide sequence of chimeric antigen selectedfrom any of the sequences shown in FIGS. 31, 35, 39, 43, 47, 51, 57, 61,65, 69, 73 and 77, wherein T may be U. For example, embodiments ofchimeric antigen nucleotides comprise, without limitation:

-   -   (a) a polynucleotide comprising or consisting of a sequence as        shown in FIG. 8 a, 11 a, 14 a, 17 a, 20 a, 23 a, 46 a, 48 a, 50        a, 52 a, 54 a or 56 a (SEQ ID NOs: 31, 35, 39, 43, 47, 51, 57,        61, 65, 69, 73 or 77), wherein T can also be U;    -   (b) a polynucleotide whose sequence is at least 80% homologous        to a sequence shown in FIG. 8 a, 11 a, 41 a, 17 a, 20 a, 23 a,        46 a, 48 a, 50 a, 52 a, 54 a or 56 a (SEQ ID NOs: 31, 35, 39,        43, 47, 51, 57, 61, 65, 69, 73 or 77);    -   (c) a polynucleotide that encodes a chimeric antigen whose        sequence encoded by a DNA contained in one of the plasmids        designated pFastBacHTa HBV S1/S2-TBD, pFastBacHTa HBV core-TBD,        pFastBacHTa HCV core(1-177)-TBD, pFastBacHTa HCV NS5A-TBD, and        pFastBacHTa HCV E2-TBD;    -   (d) a polynucleotide that encodes a chimeric antigen whose        sequence is shown in FIG. 8 b, 11 b, 14 b, 20 b, 23 b, 46 b, 48        b, 50 b, 52 b, 54 b or 56 b (SEQ ID NOs: 32, 36, 40, 44, 48, 52,        58, 62, 66, 70, 74 or 78);    -   (e) a polynucleotide that encodes a chimeric antigen-related        protein that is at least 90% identical to an entire amino acid        sequence shown in FIG. 8 b, 11 b, 14 b, 20 b, 23 b, 46 b, 48 b,        50 b, 52 b, 54 b or 56 b (SEQ ID NOs: 32, 36, 40, 44, 48, 52,        58, 62, 66, 70, 74 or 78);    -   (f) a polynucleotide that is fully complementary to a        polynucleotide of any one of (a)-(e); and    -   (g) a polynucleotide that selectively hybridizes under stringent        conditions to a polynucleotide of (a)-(f).

The invention also provides recombinant DNA or RNA molecules containinga chimeric antigen polynucleotide, an analog or homologue thereof,including but not limited to phages, plasmids, phagemids, cosmids, YACs(yeast artificial chromosomes), BACs (bacterial artificial chromosomes),as well as various viral and non-viral vectors well known in the art,and cells transformed or transfected with such recombinant DNA or RNAmolecules. Methods for generating such molecules are well known (see,for example, Sambrook et al., 1989, supra).

The invention further provides a host-vector system comprising arecombinant DNA molecule containing a chimeric antigen polynucleotide,analog or homologue thereof within a suitable prokaryotic or eukaryotichost cell. Examples of suitable eukaryotic host cells include a yeastcell, a plant cell, or an animal cell, such as a mammalian cell or aninsect cell (e.g., a baculovirus-infectible cell such as an Sf9, Sf21,Drosophila S2 or High Five™ cell). Examples of suitable mammalian cellsinclude various prostate cancer cell lines such as DU145 and TsuPr1,other transfectable or transducible prostate cancer cell lines, primarycells (PrEC), as well as a number of mammalian cells routinely used forthe expression of recombinant proteins (e.g., COS, CHO, 293, 293Tcells). More particularly, a polynucleotide comprising the codingsequence of chimeric antigen or a fragment, analog or homolog thereofcan be used to generate chimeric antigen thereof using any number ofhost-vector systems routinely used and widely known in the art.

A wide range of host-vector systems suitable for the expression ofchimeric antigens thereof are available, see for example, Sambrook etal., 1989, supra; Ausubel, Current Protocols in Molecular Biology, 1995,supra). Preferred vectors for insect cell expression include, but arenot limited to, pFastBac HTa (Invitrogen). Using such expressionvectors, chimeric antigens can be expressed in several insect celllines, including for example Sf9, Sf21, Drosophila S2 or High Five™.Alternatively, preferred yeast expression systems include Saccharomycescerevisiae, Schizosaccharomyces pombe, Pichia pastoris, and Pichiaaugust. The host-vector systems of the invention are useful for theproduction of a chimeric antigen.

A chimeric antigen or an analog or homolog thereof can be produced bycells transfected with a construct encoding a chimeric antigen. Forexample, Sf9 cells can be transfected with an expression plasmidencoding a chimeric antigen or analog or homolog thereof, the chimericantigen or related protein is expressed in the Sf9 cells, and thechimeric antigen is isolated using standard purification methods.Various other expression systems well known in the art can also beemployed. Expression constructs encoding a leader peptide joined inframe to the chimeric antigen coding sequence can be used for thegeneration of a secreted form of chimeric antigen.

As discussed herein, redundancy in the genetic code permits variation inchimeric antigen gene sequences. In particular, it is known in the artthat specific host species often have specific codon preferences, andthus one can adapt the disclosed sequence as preferred for a desiredhost. For example, preferred analog codon sequences typically have rarecodons (i.e., codons having a usage frequency of less than about 20% inknown sequences of the desired host) replaced with higher frequencycodons. Codon preferences for a specific species are calculated, forexample, by utilizing codon usage tables available on the INTERNET suchas at world wide web URL www.kazusa.or.jp/codon.

Additional sequence modifications are known to enhance proteinexpression in a cellular host. These include elimination of sequencesencoding spurious polyadenylation signals, exon/intron splice sitesignals, transposon-like repeats, and/or other such well-characterizedsequences that are deleterious to gene expression. The GC content of thesequence is adjusted to levels average for a given cellular host, ascalculated by reference to known genes expressed in the host cell. Wherepossible, the sequence is modified to avoid predicted hairpin secondarymRNA structures. Other useful modifications include the addition of atranslational initiation consensus sequence at the start of the openreading frame, as described in Kozak, Mol. Cell Biol. 9:5073-5080(1989). Skilled artisans understand that the general rule thateukaryotic ribosomes initiate translation exclusively at the 5′ proximalAUG codon is abrogated only under rare conditions (see, e.g., Kozak PNAS92(7):2662-2666 (1995) and Kozak Nucl Acids Res 15(20):8125-8148(1987)).

E. Pharmaceutical Compositions of the Invention

One aspect of the invention relates to pharmaceutical compositionscomprising a pharmaceutically acceptable excipient and a chimericantigen comprising an immune response domain and a target bindingdomain, wherein the target binding domain comprises a xenotypic antibodyfragment. In therapeutic applications, the pharmaceutical compositionscan be administered to a subject in an amount sufficient to elicit aneffective B cell, cytotoxic T lymphocyte (CTL) and/or helper Tlymphocyte (Th) response to the antigen and to prevent infenction or tocure or at least partially arrest or slow symptoms and/or complications.Amounts effective for this use will depend on, e.g., the particularcomposition administered, the manner of administration, the stage andseverity of the disease being treated, the weight and general state ofhealth of the subject, and the judgment of the prescribing physician.

The dosage for an initial therapeutic immunization (with chimericantigen) generally occurs in a unit dosage range where the lower valueis about 1, 5, 50, 500, or 1,000 ng and the higher value is about10,000; 20,000; 30,000; or 50,000 μg. Dosage values for a humantypically range from about 500 ng to about 50,000 μg per 70 kilogramsubject. Boosting dosages of between about 1.0 ng to about 50,000 μg ofchimeric antigen pursuant to a boosting regimen over weeks to months maybe administered depending upon the subject's response and condition.Administration should continue until at least clinical symptoms orlaboratory tests indicate that the condition has been prevented,arrested, slowed or eliminated and for a period thereafter. The dosages,routes of administration, and dose schedules are adjusted in accordancewith methodologies known in the art.

A human unit dose form of a chimeric antigen is typically included in apharmaceutical composition that comprises a human unit dose of anacceptable carrier, in one embodiment an aqueous carrier, and isadministered in a volume/quantity that is known by those of skill in theart to be useful for administration of such polypeptides to humans (see,e.g., Remington: The Science and Practice of Pharmacy, 20^(th) Edition,A. Gennaro, Editor, Lippincott Williams & Wilkins, Baltimore, Md.,2000). As appreciated by those of skill in the art, various factors caninfluence the ideal dose in a particular case. Such factors include, forexample, half life of the chimeric antigen, the binding affinity of thechimeric antigen, the immunogenicity of the composition, the desiredsteady-state concentration level, route of administration, frequency oftreatment, and the influence of other agents used in combination withthe treatment method of the invention, as well as the health status of aparticular subject.

In certain embodiments, the compositions of the present invention areemployed in serious disease states, that is, life-threatening orpotentially life-threatening situations. In such cases, as a result ofthe relative nontoxic nature of the chimeric antigen in preferredcompositions of the invention, it is possible and may be felt desirableby the treating physician to administer substantial excesses of thesechimeric antigens relative to these stated dosage amounts.

The concentration of chimeric antigen of the invention in thepharmaceutical formulations can vary widely, i.e., from less than about0.1%, usually at or at least about 2% to as much as 20% to 50% or moreby weight, and will be selected primarily by fluid volumes, viscosities,etc., in accordance with the particular mode of administration selected.

The pharmaceutical compositions can be delivered via any route known inthe art, such as parenterally, intrathecally, intravascularly,intravenously, intramuscularly, transdermally, intradermally,subcutaneously, intranasally, topically, orally, rectally, vaginally,pulmonarily or intraperitoneally. Preferably, the composition isdelivered by parenteral routes, such as subcutaneous or intradermaladministration.

The pharmaceutical compositions can be prepared by mixing the desiredchimeric antigens with an appropriate vehicle suitable for the intendedroute of administration. In making the pharmaceutical compositions ofthis invention, the chimeric antigen is usually mixed with an excipient,diluted by an excipient or enclosed within a carrier that can be in theform of a capsule, sachet, paper or other container. When thepharmaceutically acceptable excipient serves as a diluent, it can be asolid, semi-solid, or liquid material, which acts as a vehicle, carrieror medium for the therapeutic agent. Thus, the compositions can be inthe form of tablets, pills, powders, lozenges, sachets, cachets,elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solidor in a liquid medium), ointments containing, for example, up to 10% byweight of the chimeric antigen, soft and hard gelatin capsules,suppositories, sterile injectable solutions, and sterile packagedpowders.

Some examples of suitable excipients include, but are not limited to,dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calciumphosphate, alginates, tragacanth, gelatin, calcium silicate,microcrystalline cellulose, polyvinylpyrrolidone, cellulose, sterilewater, syrup, and methyl cellulose. The formulations can additionallyinclude: lubricating agents such as talc, magnesium stearate, andmineral oil; wetting agents; emulsifying and suspending agents;preserving agents such as methyl- and propylhydroxy-benzoates;sweetening agents; and flavoring agents. The compositions of theinvention can be formulated so as to provide quick, sustained or delayedrelease of the chimeric antigen after administration to the subject byemploying procedures known in the art. See, e.g., Remington, supra, atpages 903-92 and pages 1015-1050.

For preparing solid compositions such as tablets, the chimeric antigenis mixed with a pharmaceutical excipient to form a solid preformulationcomposition containing a homogeneous mixture of a chimeric antigen ofthe present invention. When referring to these preformulationcompositions as homogeneous, it is meant that the chimeric antigen isdispersed evenly throughout the composition so that the composition maybe readily subdivided into equally effective unit dosage forms such astablets, pills and capsules.

The tablets or pills of the present invention may be coated or otherwisecompounded to provide a dosage form affording the advantage of prolongedaction. For example, the tablet or pill can comprise an inner dosage andan outer dosage component, the latter being in the form of an envelopeover the former. The two components can be separated by an entericlayer, which serves to resist disintegration in the stomach and permitthe inner component to pass intact into the duodenum or to be delayed inrelease. A variety of materials can be used for such enteric layers orcoatings, such materials including a number of polymeric acids andmixtures of polymeric acids with such materials as shellac, cetylalcohol, and cellulose acetate.

The liquid forms in which the novel compositions of the presentinvention may be incorporated for administration orally or by injectioninclude aqueous solutions, suitably flavored syrups, aqueous or oilsuspensions, and flavored emulsions with edible oils such as corn oil,cottonseed oil, sesame oil, coconut oil, or peanut oil, as well aselixirs and similar pharmaceutical vehicles.

In preparing a composition for parenteral administration strictattention must be paid to tonicity adjustment to reduce irritation. Areconstitutable composition is a sterile solid packaged in a dry form. Areconstitutable composition is preferred because it is more stable whenstored as a dry solid rather than in a solution ready for immediateadministration. The dry solid is usually packaged in a sterile containerwith a butyl rubber closure to ensure the solid is kept at an optimalmoisture range. A reconstitutable dry solid is formed by dry fill, spraydrying, or freeze-drying methods. Descriptions of these methods may befound, e.g., in Remington, supra, at pages 681-685 and 802-803.

Compositions for parenteral injection are generally dilute, and thecomponent present in the higher proportion is the vehicle. The vehiclenormally has no therapeutic activity and is nontoxic, but presents thechimeric antigen to the body tissues in a form appropriate forabsorption. Absorption normally will occur most rapidly and completelywhen the chimeric antigen is presented as an aqueous solution. However,modification of the vehicle with water-miscible liquids or substitutionwith water-immiscible liquids can affect the rate of absorption.Preferably, the vehicle of greatest value for this composition isisotonic saline. In preparing the compositions that are suitable forinjection, one can use aqueous vehicles, water-miscible vehicles, andnonaqueous vehicles

Additional substances may be included in the injectable compositions ofthis invention to improve or safeguard the quality of the composition.Thus, an added substance may affect solubility, provide for subjectcomfort, enhance the chemical stability, or protect the preparationagainst the growth of microorganisms. Thus, the composition may includean appropriate solubilizer, substances to act as antioxidants, andsubstances that act as a preservative to prevent the growth ofmicroorganisms. These substances will be present in an amount that isappropriate for their function, but will not adversely affect the actionof the composition. Examples of appropriate antimicrobial agents includethimerosal, benzethonium chloride, benzalkonium chloride, phenol, methylp-hydroxybenzoate, and propyl p-hyrodxybenzoate. Appropriateantioxidants may be found in Remington, supra, at p. 1015-1017.

In certain embodiments, liposomes, nanocapsules, microparticles, lipidparticles, vesicles, and the like, are used for the administration ofthe chimeric antigens of the present invention. In particular, thecompositions of the present invention may be formulated for deliveryeither encapsulated in a lipid particle, a liposome, a vesicle, ananosphere, or a nanoparticle or the like. Alternatively, compositionsof the present invention can be bound, either covalently ornon-covalently, to the surface of such carrier vehicles.

Compositions administered via liposomes may also serve: 1) to target thechimeric antigen to a particular tissue, such as lymphoid tissue; 2) totarget selectively to antigen presenting cells; or, 3) to increase thehalf-life of the peptide composition. Liposomes include emulsions,foams, micelles, insoluble monolayers, liquid crystals, phospholipiddispersions, lamellar layers and the like. In these preparations, thechimeric antigen to be delivered is incorporated as part of a liposome,alone or in conjunction with a molecule that binds to a receptorprevalent among lymphoid cells, such as monoclonal antibodies that bindto the CD45 antigen, or with other therapeutic or immunogeniccompositions. Thus, liposomes either filled or decorated with a desiredchimeric antigen of the invention can be directed to the site oflymphoid cells, where the liposomes then deliver the chimeric antigens.Liposomes for use in accordance with the invention are formed fromstandard vesicle-forming lipids, which generally include neutral andnegatively charged phospholipids and a sterol, such as cholesterol. Theselection of lipids is generally guided by consideration of, e.g.,liposome size, acid lability and stability of the liposomes in the bloodstream. A variety of methods are available for preparing liposomes, asdescribed in, e.g., Szoka, et al., Ann. Rev. Biophys. Bioeng. 9:467-508(1980), and U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and5,019,369. A liposome suspension containing a chimeric antigen may beadministered intravenously, locally, topically, etc. in a dose whichvaries according to, inter alia, the manner of administration, thechimeric antigen being delivered, and the stage of the disease beingtreated.

Compositions for inhalation or insufflation include solutions andsuspensions in pharmaceutically acceptable, aqueous or organic solvents,or mixtures thereof, and powders. The liquid or solid compositions maycontain suitable pharmaceutically acceptable excipients as describedherein. The compositions can be administered by the oral or nasalrespiratory route for local or systemic effect. Compositions inpharmaceutically acceptable solvents may be nebulized by use of inertgases. Nebulized solutions may be inhaled directly from the nebulizingdevice or the nebulizing device may be attached to a facemask tent, orintermittent positive pressure breathing machine. Solution, suspension,or powder compositions may be administered, preferably orally ornasally, from devices that deliver the formulation in an appropriatemanner.

Another formulation employed in the methods of the present inventionemploys transdermal delivery devices (“patches”). Such transdermalpatches may be used to provide continuous or discontinuous infusion ofthe chimeric antigen of the present invention in controlled amounts. Theconstruction and use of transdermal patches for the delivery ofpharmaceutical agents is well known in the art. See, for example, U.S.Pat. No. 5,023,252, herein incorporated by reference. Such patches maybe constructed for continuous, pulsatile, or on demand delivery ofpharmaceutical agents.

Additionally, it may be advantageous to include at least one antiviraltherapeutic or chemotherapeutic in addition to the chimeric antigen andpharmaceutical excipient. Antiviral therapeutics include, but are notlimited to, peptidomimetics (such as amprenavir, indinavir, lopinavir,nelfinavir, ritonavir, and saquinavir), polynucleotides (such asampligen and fomivirsen), purine/pyrimidinones (such as abacavir,acyclovir, adefovir, cidofovir, cytarabine, didanosine,dideoxyadenosine, dipivoxil, edoxudine, emtricitabine, entecovir,famciclovir, ganciclovir, idoxuridine, inosine pranobex, lamivudine,MADU, penciclovir, sorivudine, stavudine, tenofovir, trifluridine,valacyclovir, valganciclovir, vidarabine, zalcitabine, and zidovudine),sialic acid analogs (such as oseltamivir and zanamivir), acemannan,acetylleucine monoethanolamine, amantadine, amidinomycin, ateviridine,capravirine, delavirdine, n-docosanol, efavirenz, foscarnet sodium,interferon-α, interferon-β, interferon-γ, kethoxal, lysozyme,methisazone, moroxydine, nevirapine, pentafuside, pleconaril,podophyllotoxin, ribavirin, rimantidine, stallimycin, statolon,termacamra, and traomantadine. Other appropriate antiviral agents arediscussed in Remington: supra, at Chapter 87: Anti-Infectives, pp.1507-1561, particularly pp. 1555-1560. Preferred antiviral therapeuticsfor inclusion in the pharmaceutical compositions of the presentinvention include adefovir, dipivoxil, entecovir, lamivudine andribavirin.

In some embodiments it may be desirable to include in the pharmaceuticalcompositions of the invention at least one component which primesB-Lymphocytes or T lymphocytes. Lipids have been identified as agentscapable of priming CTL in vivo. For example, palmitic acid residues canbe attached to the ε- and α-amino groups of a lysine residue and thenlinked, e.g., via one or more linking residues such as Gly, Gly-Gly-,Ser, Ser-Ser, or the like, to an immunogenic peptide. The lipidatedpeptide can then be administered either directly in a micelle orparticle, incorporated into a liposome, or emulsified in an adjuvant,e.g., incomplete Freund's adjuvant. In a preferred embodiment, aparticularly effective immunogenic composition comprises palmitic acidattached to s- and α-amino groups of Lys, which is attached via linkage,e.g., Ser-Ser, to the amino terminus of the immunogenic peptide.

As another example of lipid priming of CTL responses, E. colilipoproteins, such as tripalmitoyl-S-glycerylcysteinlyseryl-serine(P₃CSS) can be used to prime virus specific CTL when covalently attachedto an appropriate peptide (see, e.g., Deres, et al., Nature 342:561(1989)). Chimeric antigens of the invention can be coupled to P₃CSS, forexample, and the lipopeptide administered to an individual tospecifically prime an immune response to the target antigen.

While the compositions of the present invention should not require theuse of adjuvants, adjuvant can be used. Various adjuvants may be used toincrease the immunological response, depending on the host species, andincluding but not limited to Freund's (complete and incomplete), mineralgels such as aluminum hydroxide, surface active substances such aslysolecithin, detergents, pluronic polyols, polyanions, peptides, oilemulsions, keyhole limpet hemocyanins, dinitrophenol, immunostimulatorypolynucleotide sequences, and potentially useful human adjuvants such asBCG (bacille Calmette-Guerin) and corynebacterium parvum. Additionaladjuvants are also well known in the art.

F. Methods of Using Chimeric Antigens

Another aspect of the invention provides methods of enhancing antigenpresentation in antigen presenting cells, said method comprisingadministering, to the antigen presenting cells, a chimeric antigen thatcomprises an immune response domain and a target binding domain, whereinthe target binding domain comprises a xenotypic antibody fragment. In apreferred embodiment, the antigen presenting cells are dendritic cells.

An aspect of the invention relates to methods of activating antigenpresenting cells comprising contacting the antigen presenting cell witha chimeric antigen that comprises an immune response domain and a targetbinding domain, wherein the target binding domain comprises a xenotypicantibody fragment. In a preferred embodiment, the antigen presentingcell is contacted with the chimeric antigen in vivo. In anotherpreferred embodiment, the contacting takes place in a human.

Yet another aspect of the invention provides methods of eliciting animmune response, said method comprising administering to an animal achimeric antigen that comprises an immune response domain and a targetbinding domain, wherein the target binding domain comprises a xenotypicantibody fragment. The immune response can be a humoral and/or cellularimmune response. In a preferred embodiment, the cellular immune responseis both a Th1 and a Th2 response.

Another aspect of the invention provides methods of treatingimmune-treatable conditions comprising administering, to an animal inneed thereof, a chimeric antigen that comprises an immune responsedomain and a target binding domain, wherein the target binding domaincomprises a xenotypic antibody fragment. Preferably, theimmune-treatable condition is a viral infection or cancer. Morepreferably, the immune-treatable condition is a chronic viral infection.Most preferably, the immune-treatable condition is a chronic hepatitis Bviral infection or a chronic hepatitis C viral infection. For thetreatment of HBV, preferably the immune response domain comprises aprotein selected from the group consisting of a HBV Core protein, a HBVS protein, a HBV S1 protein, a HBV S2 protein, and combinations thereof.For the treatment of HCV, preferably the immune response domaincomprises a protein selected from the group consisting of a HCV Core(1-191) protein, a HCV Core (1-177) protein, a HCV E1 protein, a HCV E2protein, a HCV E1-E2 protein, a HCV NS3A protein, a HCV NS5A protein,and combinations thereof.

Another aspect of the invention provides methods of vaccinating ananimal against a viral infection comprising administering to the animala chimeric antigen that comprises an immune response domain and a targetbinding domain, wherein the target binding domain comprises a xenotypicantibody fragment. The method of the invention can prophylactically ortherapeutically vaccinate the animal against the viral infection.

The present invention also comprises methods of using the compositionsof the present invention to bind and activate antigen presenting cells,such as dendritic cells. The present invention also comprises methods ofusing the compositions of the present invention to activate T cells. Thepresent invention also comprises a method of delivering an antigen to animmune system cell, such as an antigen presenting cell. The presentinvention also comprises compositions and methods for activating ahumoral and/or cellular immune response in an animal or human, saidmethod comprising administering one or more chimeric antigens of thepresent invention.

Following cloning and expression, the chimeric antigen is evaluated forits efficacy in generating an immune response. Evaluation involvespresenting the chimeric antigen to dendritic cells ex vivo or in vivo.The dendritic cells are presented to T-lymphocytes and evaluated for theproduction of interferon-γ as a marker of T cell response. Specifically,in the ex vivo situation, naive dendritic cells are isolated fromperipheral blood. Dendritic cells process and present antigen to naiveT-lymphocytes. The chimeric antigen is then presented to naive dendriticcells for processing. These stimulated dendritic cells are in turnpresented to a naive T cells, which cause their activation into effectorcells, e.g. helper T cells or cytotoxic T-lymphocytes. Activation of theT cells by the dendritic cells is then evaluated by measuring markers,e.g. interferon-γ levels, by a known procedure (Berlyn, et al., Clin.Immunol 101(3):276-283 (2001)). An increase in the percentage of T cellsthat secrete interferon-γ by at least 50% over background predictsefficacy in vivo. In preferred embodiments, the percentage increase isat least 55%, 60%, 65%, 70%, 75%, 80%, 90% or 100%. In the case of thein vivo situation, the chimeric antigen is directly introducedparenterally in the host, where available dendritic and otherantigen-processing cells have the capacity to interact with all antigensand process them accordingly.

G. Combination Therapy

Another aspect of the invention provides compositions for treating viralinfections comprising a chimeric antigen and an antiviral agent. Theinvention also provides methods of treating viral infections comprisingadministering a chimeric antigen and an antiviral agent, eitherconcurrently or sequentially.

Chimeric antigens have been shown to induce specific anti-HBV S1/S2cytotoxic T cell functions ex vivo, to induce anti-HBV S1/S2 humoralresponses in mice, and transiently reduce the viral load in ducksinfected with the hepatitis B duck virus (DHBV). The use of a chimericantigen in combination with an antiviral agent, such as a nucleosideanalogue, may prove to be highly efficacious in inducing sustainedresponses in the treatment of subjects suffering from chronic hepatitisB. The mechanisms of action of the two agents used in combination mayproduce synergistic effects in treatment of hepatitis B subjects. Whilenot being limited to a particular therapy, a nucleoside analogue, forexample, would reduce the number of viral particles circulating in theblood and hence reduce the antigenic load that the immune system musteliminate, and the chimeric antigen would induce a highly specificcellular immune response that would eliminate cells that harbor virus,viral antigens and viral DNA/RNA. In addition, the chimeric antigenwould induce a humoral immune response that would neutralize and removecirculating viral particles. Furthermore, the immune mechanism of actionof the chimeric antigen could also minimize the toxicity of antiviralagents by permitting lower doses of the antiviral agent to beadministered over a shorter period of time. A reduction in the length oftime to achieve a sustained response may reduce the chances ofdevelopment of drug-resistant viral mutants normally induced byantiviral agents, especially nucleoside analogue antiviral agents, whenused alone in long-term therapy.

In brief, combination therapy with the hepatitis B chimeric antigen(e.g. S1/S2-TBD) and a nucleoside analogue in the treatment of hepatitisB has the potential to effect a complete cure of chronic HBV infection.Likewise, a combination of an HCV antiviral such as ribavirin along withthe HCV chimeric antigens described herein will produce antigen-specificcellular as well as humoral immune response and thus clear HCV infectionin chronically infected subjects.

H. Methods of Preparation

One aspect of the invention provides methods for producing a chimericantigen comprising (a) providing a microorganism or cell line,preferably a eukaryotic, more preferably, a non-mammalian microorganismor cell line, that comprises a polynucleotide encoding a chimericantigen; and (b) culturing said microorganism or cell line underconditions whereby the chimeric antigen is expressed. Preferably, themicroorganism or cell line is a yeast, a plant cell line or an insectcell line. More preferably, the cell line is an insect cell lineselected from the group consisting of Sf9, Sf21, Drosophila S2, and HighFive™.

The present invention uses established recombinant DNA technology forproducing the fusion proteins of selected antigen(s) and the TBD thatare necessary in the practice of the invention. Fusion proteinconstructs are generated at the DNA level incorporating specificrestriction enzyme sites, which are exploited in incorporating thedesired DNA fragment into expression vectors, and used to express thedesired fusion proteins in a heterologous expression system. As usedherein, the term “vector” denotes plasmids that are capable of carryingthe DNA, which encode the desired protein(s). The plasmid vectors usedin the present invention include, but are not limited to, pFastBac HTaand the corresponding recombinant “BACMIDS” generated in DH10Bac™ E.coli (Invitrogen). It is possible to mobilize the ORF of the desiredproteins and produce other recombinant plasmids for expression of theproteins in other systems, (bacterial or mammalian), in addition to theBac-To-Bac™ baculovirus expression system (Invitrogen), employed in thepresent invention. The term “expression” is used to mean thetranscription of the DNA sequence into mRNA, the translation of the mRNAtranscript into the fusion protein.

This is achieved by the transposition of the gene of interest into thebacmids, transfected into Sf9 insect cells and recombinant baculovirusproduced. These are used to infect Sf9 or High Five™ insect cells, whichproduce the protein of interest. All the recombinant proteins producedhave an N-terminal 6×His tag, which is exploited in the purification ofthe proteins by using Ni-NTA Agarose (Qiagen). The proteins also have anN-terminal rTEV protease cleavage site cloned in. The Ni-purifiedprotein is subjected to digestion with rTEV protease (Invitrogen), whichalso has an N-terminal 6×His tag. Following the protease digestion, themixture can be loaded on to a Ni-NTA agarose column and the pure proteincan be eluted out, while the 6×His tagged fragments will be bound to thecolumn. This method of purification is standard procedure and oneskilled in the art would be able to understand the methodology withoutfurther explanation.

Cloning and expression of the DNA sequences which encode the viralantigen and the Fc fragment of the murine monoclonal antibody togenerate the chimeric antigen can be achieved through two approaches.The first approach involves cloning the two proteins as a fusionprotein, while the second approach involves incorporating specific“bio-linkers” such as biotin or streptavidin in either of the molecules,purifying them separately and generating the chimeric antigen.

In an exemplary embodiment, a monoclonal antibody (2C12) was generatedagainst the Hepatitis B virus surface antigen, and the hybridoma, whichproduced this monoclonal antibody, was used to isolate the total RNA forthe murine immunoglobulin G. Total RNA was isolated and used to clonethe murine Fc fragment. Specifically, the total RNA from a hybridomacell that expresses murine IgG is isolated using Trizol® reagent(Invitrogen/Gibco BRL, product catalog number 10551-018, 10298-016; amonophasic solution of phenol and guanidine isothiocyante, as describedin U.S. Pat. No. 5,346,994). The mRNA was purified from total RNA byaffinity chromatography on an oligo-dT column (Invitrogen/Gibco BRL,product catalog number 15939-010). A complementary DNA (cDNA) wasproduced using reverse transcriptase in a polymerase chain reaction. Theoligonucleotide primers were designed to add unique restriction enzymerecognition sites to facilitate cloning. This cDNA was cloned using theBac-To-Bac™ baculovirus expression system (Invitrogen/Gibco BRL, productcatalog number 15939-010).

The baculovirus system, preferentially, is used because not only arelarge amounts of heterologous proteins are produced, but also becausepost-translational modifications, such as phosphorylation andglycosylation, of eukaryotic proteins occur within the infected insectcell. In this expression system, the DNA can be cloned into vectorscalled pFastBac™ as illustrated schematically in FIG. 4(Invitrogen/Gibco BRL, product catalog number 15939-010). In theBac-To-Bac™ system, the generation of recombinants is based onsite-specific transposition with the bacterial transposon Tn7. The geneof interest is cloned into pFastBac™, which has mini-Tn7 elementsflanking the cloning sites. The plasmid is transformed into Escherichiacoli strain DH10Bac™ (Invitrogen/Gibco BRL, product catalog number10361-012), which has a baculovirus shuttle plasmid (bacmid) containingthe attachment site of Tn7 within a LacZ gene. Transposition disruptsthe LacZ gene so that only recombinants produce white colonies and areeasily selected for. The advantage of using transposition in E. coli isthat single colonies contain only recombinants so that plaquepurification and screening are not required. The recombinant bacmids aretransfected in insect cells to generate baculoviruses that expressrecombinant proteins.

The Bac-To-Bac™ baculovirus expression system is commercially availablefrom Invitrogen and the procedures used were as described in the companyprotocols, available, for example, at www.invitrogen.com. The gene ofinterest is cloned into pFastBac HTa donor plasmid and the production ofrecombinant proteins is based upon the Bac-To-Bac™ baculovirusexpression system (Invitrogen).

In the next step, the pFastBac HTa donor plasmid containing the gene ofinterest is used in a site-specific transposition in order to transferthe cloned gene into a baculovirus shuttle vector (bacmid). This isaccomplished in E. coli strain DH10Bac™. The DH10Bac™ cells contain thebacmid, which confers kanamycin resistance and a helper plasmid, whichencodes the transposase and confers resistance to tetracycline. Therecombinant pFastBac HTa plasmids with the gene of interest aretransformed into DH10Bac™ cells for the transposition to generaterecombinant bacmids. A 100 μl aliquot of competent DH10Bac™ cells isthawed on ice, the pFastBac HTa based plasmids are added and the mixtureis incubated on ice for 30 minutes. The mixture is given a heat shockfor 45 seconds at 42° C. and then chilled on ice for 2 minutes. Themixture is then added to 900 μL of LB media and incubated for 4 hours at37° C. The transformed cells are serially diluted with LB to 10⁻¹ and10⁻² and 100 μl of each dilution is plated on Luria broth (LB) agarplates (supplemented with 50 μg/ml kanamycin, 7 μg/ml gentamicin, 10μg/ml tetracycline, 100 μg/ml X-gal, and 40 μg/ml IPTG) and incubatedfor at least 36 hours at 37° C. The gentamicin resistance is conferredby the pFastBac HTa and the X-gal and IPTG are used to differentiatebetween white colonies (recombinant bacmids) from blue colonies (nonrecombinant). The white colonies are picked and inoculated into 2 ml ofLB (supplemented with 50 μg/ml kanamycin, 7 μg/ml gentamicin and 10μg/ml tetracycline) and incubated overnight at 37° C., with shaking. Asterile loop is used to sample a small amount of the overnight cultureand the sample is streaked onto a fresh LB agar plate (supplemented with50 μg/ml kanamycin, 7 μg/ml gentamicin, 10 μg/ml tetracycline, 100 μg/mlX-gal, and 40 μg/ml isopropylthio-β-D-galactoside (IPTG)) and incubatedfor at least 36 hours at 37° C. to confirm a white phenotype.

Recombinant bacmids were isolated by standard protocols (Sambrook,supra); the DNA sample was dissolved in 40 μl of TE (10 mM Tris-HCl pH8, 1 mM EDTA (ethylenediaminetetraacetic acid)) and used fortransfections.

In order to produce baculoviruses, the bacmid is transfected into Sf9insect cells. Sf9 cells (9×10⁵) were seeded into each well of a 6-wellcell culture dish (35 mm wells) in 2 ml of ESF 921 (Expression Systems)and allowed to attach for at least 1 hour at 27° C. Transfections werecarried out using Cellfectin® Reagent (Invitrogen, Cat. No. 10362-010; a1:1.5 (M/M) liposome formulation of the cationic lipid N,N^(I),N^(II),N^(III)-Tetramethyl-N,N^(I), N^(II), N^(III)-tetrapalmitylspermine anddioleoyl phosphatidylethanolammine in membrane filtered water) as perthe protocols provided by the supplier of the Sf9 cells. Followingtransfection, the cells were incubated at 27° C. for 72 hours. Themedium containing baculovirus was collected and stored at 4° C. in thedark.

The efficiency of the transfection was verified by checking forproduction of baculoviral DNA. The isolated baculovirus DNA is subjectedto PCR to screen for the inserted gene of interest. The primers used arepFastBac HTa 5′ (sense) TATTCCGGATTATTCATACCG (SEQ ID NO: 3) andpFastBac HTa 3′ (antisense) 5′ CTCTACAAATGTGGTATGGC (SEQ ID NO: 4).Amplified products were separated on an agarose gel (0.8%). Theexpression of the heterologous protein in the cells was verified by SDSpolyacrylamide gel electrophoresis (SDS-PAGE) and Western blots usingthe 6×His tag monoclonal antibody (Clontech) as the probe.

Once production of baculovirus and the expression of protein have beenconfirmed, the virus stock is amplified to produce a concentrated stockof the baculovirus that carry the gene of interest. It is standardpractice in the art to amplify the baculovirus at least two times, andin all protocols described herein this standard practice was adhered to.After the second round of amplification, the concentration of thegenerated baculovirus was quantified using a plaque assay according tothe protocols described by the manufacturer of the kit (Invitrogen). Themost appropriate concentration of the virus to infect insect cells andthe optimum time point for the production of the desired protein wasalso established.

The DNA encoding proteins of interest are generated by PCR witholigonucleotide primers bearing unique restriction enzyme sites fromplasmids that contain a copy of the entire viral genome and cloned withthe Fc DNA as a fusion protein. This chimeric protein is purified byprotein A or G affinity chromatography using techniques known to thoseskilled in the art.

The second approach for linking the IRD and TBD involves incorporatingspecific “bio-linkers” such as biotin or streptavidin in either of themolecules, purifying them separately and generating the chimericantigen. The viral antigens of interest are cloned into plasmids thatcontrol the expression of proteins by the bacteriophage T7 promoter. Therecombinant plasmid is then transformed into an E. coli strain, e.g.BL21(DE3) Codon Plus™ RIL cells (Stratagene, product catalog number230245), which has production of T7 RNA polymerase regulated by the lacrepressor. The T7 RNA polymerase is highly specific for T7 promoters andis much more processive (˜8 fold faster) than the E. coli host's RNApolymerase. When production of T7 RNA polymerase is induced byisopropylthio-β-D-galactoside (IPTG), the specificity and processivityof T7 RNA polymerase results in a high level of transcription of genesunder control of the T7 promoter. In order to couple two proteinstogether, the tight binding between biotin and streptavidin isexploited. In E. coli, the BirA enzyme catalyzes the covalent linkage ofbiotin to a defined lysine residue in a specific recognition sequence.The murine Fc fragment is expressed in the baculovirus system, asdescribed above, as a fusion protein with streptavidin. These twoproteins can be mixed to form a dimeric protein complex bybiotin-streptavidin binding.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology, microbiology,recombinant DNA, and immunology, which are within the skill of the art.Such techniques are explained fully in the literature. See e.g.,Sambrook, supra; and Ausubel, supra.

I. Article of Manufacture

Another aspect of this invention provides an article of manufacture thatcomprises a container holding a composition, comprising a chimericantigen, that is suitable for injection or reconstitution for injectionin combination with printed labeling instructions providing a discussionof how to administer the composition parenterally, e.g. subcutaneously,intramuscularly, intradermally, nasally or intravascularly. Thecomposition will be contained in any suitable container that will notsignificantly interact with the composition and will be labeled with theappropriate labeling that indicates it will be for parenteral use.Associated with the container will be the labeling instructionsconsistent with the method of treatment as described hereinbefore. Thecontainer that holds the composition of this invention may be acontainer having a liquid composition suitable for injection that has anappropriate needle for injection and a syringe so that the patient,doctor, nurse, or other practitioner can administer the chimericantigen. Alternatively, the composition may be a dry or concentratedcomposition containing a soluble version of the chimeric antigen, to becombined or diluted with an aqueous or nonaqueous vehicle to dissolve orsuspend the composition. Alternatively, the container may have asuspension in a liquid or may be an insoluble version of the salt forcombination with a vehicle in which the insoluble version will besuspended. Appropriate containers are discussed in Remington, supra,pages 788-789, 805, 850-851 and 1005-1014

The kit of the invention will typically comprise the container describedabove and one or more other containers comprising materials desirablefrom a commercial and user standpoint, including buffers, diluents,filters, needles, syringes, and package inserts with instructions foruse. A label can be present on the container to indicate that thecomposition is used for a specific therapy or non-therapeuticapplication, and can also indicate directions for either in vivo or exvivo use, such as those described above. Directions and or otherinformation can also be included on an insert which is included with thekit

V. EXAMPLES

The following non-limiting examples provide further illustration of theinvention.

Example 1 Construction of Murine TBD Protein Expression Vector

The mouse IgG1 DNA sequences encoding amino acids of a portion ofC_(H)1-Hinge-C_(H)2-C_(H)3 region was generated from mRNA isolated fromthe hybridoma (2C12), which produces mAb against HBV surface antigen(sAg). Total RNA was isolated from 2C12 using Trizol® reagent (Gibco BRLcat. No. 15596-026) and the DNA of the TBD was generated by RT-PCR usingSuperscript First-strand Synthesis (Invitrogen Cat. No. 11904-018). ThePCR primers contained linker sequences encoding the linkerpeptide—SRPQGGGS—(SEQ ID NO: 28) at the 5′ terminus, a unique Not I siteat the 5′ and a unique Hind III restriction site at the 3′ end. Theresulting cDNA contains (5′ Not I)-linker sequence-C_(h)1(VDKKI)-C_(H)2-C_(H)3-(3′ Hind III). Following digestion with therespective enzymes, the fragment is ligated with pFastBac HTa expressionvector plasmid (Invitrogen) using the same restriction enzyme sites. The5′ primer used for PCR amplification was (Sense) 5′TGTCATTCTGCGGCCGCAAGGCGGCGGATCCGTGGACAAGAAAATTGTGCCC AGG (SEQ ID NO: 1)and the 3′ primer was (antisense) 5′ACGAATCAAGCTTTGCAGCCCAGGAGAGTGGGAGAG (SEQ ID NO: 2), which contained theNot I and Hind III sites, respectively. The following protocol was usedfor directional cloning. The generated fragment was digested with therespective enzymes, purified on agarose gel and cloned into the vectorplasmid. The DNA sequence and the correctness of the ORF were verifiedby standard sequencing methods.

Following the cloning of the gene of interest (e.g. TBD) into thepFastBac HTa donor plasmid, the production of recombinant proteins wasbased upon the Bac-To-Bac™ baculovirus expression system (Invitrogen).The next step was site-specific transposition of the cloned gene into abaculovirus shuttle vector (Bacmid). This was accomplished in a strainof E. coli called DH10Bac™. The DH10Bac™ cells contain the bacmid, whichconfers kanamycin resistance and a helper plasmid, which encodes thetransposase and confers resistance to tetracycline. The recombinantpFastBac HTa plasmids with the gene of interest (TBD) were transformedinto DH10Bac™ cells for the transposition to generate recombinantbacmids. A 100 μl aliquot of competent DH10Bac™ cells was thawed on ice,the pFastBac HTa based plasmids were added and the mixture was incubatedon ice for 30 minutes. The mixture was given a heat shock for 45 secondsat 42° C. and then chilled on ice for 2 minutes. The mixture was thenadded to 900 μL of LB media and incubated for 4 hours at 37° C. Thetransformed cells were serially diluted with LB to 10⁻¹ and 10⁻² and 100μl of each dilution was plated on LB agar plates supplemented with 50μg/ml kanamycin, 7 μg/ml gentamicin, 10 μg/ml tetracycline, 100 μg/mlX-gal, and 40 μg/ml IPTG and incubated for at least 36 hours at 37° C.The gentamicin resistance was conferred by the pFastBac HTa and theX-gal and IPTG were used to differentiate between white colonies(recombinant bacmids) from blue colonies (non recombinant). The whitecolonies were picked and inoculated into 2 ml of LB supplemented with 50μg/ml kanamycin, 7 μg/ml gentamicin and 10 μg/ml tetracycline andincubated overnight at 37° C., with shaking. A sterile loop was used tosample a small amount of the overnight culture and the sample wasstreaked onto a fresh LB agar plate supplemented with 50 μg/mlkanamycin, 7 μg/ml gentamicin, 10 μg/ml tetracycline, 100 μg/ml X-gal,and 40 μg/ml IPTG and incubated for at least 36 hours at 37° C. toconfirm a white phenotype.

Recombinant bacmids were isolated by standard protocols (Sambrook,supra). The DNA sample was dissolved in 40 μl of TE (10 mM Tris-HCl pH8, 1 mM EDTA) and used for transfections.

In order to produce baculoviruses, the bacmid was transfected into Sf9insect cells. Sf9 cells (9×10⁵) were seeded into each well of a 6-wellcell culture dish (35 mm wells) in 2 ml of ESF 921 (Expression Systems)and allowed to attach for at least 1 hour at 27° C. Transfections werecarried out using Cellfectin® Reagent (Invitrogen, Cat. No. 10362-010)as per the protocols provided by the supplier of the Sf9 cells.Following transfection, the cells were incubated at 27° C. for 72 hours.The medium containing baculovirus was collected and stored at 4° C. inthe dark.

The efficiency of the transfection was verified by checking forproduction of baculoviral DNA. The isolated baculovirus DNA wassubjected to PCR to screen for the inserted gene of interest (TBD). Theprimers used are pFastBac HTa 5′ (sense) TATTCCGGATTATTCATACCG (SEQ IDNO: 3) and pFastBac HTa 3′ (antisense) 5′ CTCTACAAATGTGGTATGGC (SEQ IDNO: 4). Amplified products were visualized on an agarose gel (0.8%). Theexpression of the heterologous protein in the cells was verified by SDSpolyacrylamide gel electrophoresis (SDS-PAGE) and Western blots usingthe 6×His tag monoclonal antibody (Clonetech) as the probe.

Once production of baculovirus and the expression of protein had beenconfirmed, the virus production was amplified to produce a concentratedstock of the baculovirus that carry the gene of interest (e.g. TBD). Itis standard practice in the art to amplify the baculovirus at least twotimes, and in all protocols described herein this standard practice wasadhered to. After the second round of amplification, the concentrationof the generated baculovirus was quantified using a plaque assayaccording to the protocols described by the manufacturer of the kit(Invitrogen). The most appropriate concentration of the virus to infectSf9 and High Five™ cells and the optimum time point for the productionof the desired protein was established as well.

Example 2 Construction of HBV Surface Antigen S1/S2 and HBV S1/S2-TBDFusion Protein Expression Vectors

The DNA encoding the HBV sAg fragment S1/S2 was generated from theplasmid pRSetB HBV S1/S2 template using PCR methodology. The primersused were: (sense) 5′ GGATCTGTACGACGATGACG (SEQ ID NO: 5) and the 3′primer (antisense) 5′ AGTCATTCTGCGGCCGCGAGTTCGTCACAGGGTCCCCGG (SEQ IDNO: 6) containing the restriction enzyme site Not I. The 5′ endcontained a unique Bam HI site derived from the parent plasmid that wasused for ligations. Amplified DNA was digested with Bam HI/Not I andligated with pFastBac HTa expression vector to generate the expressionplasmid for HBV S1/S2 protein. The fragment was ligated with the plasmidpFastBac HTa-TBD (described in example 1) following the digestion withthe respective enzymes. This produced the expression plasmid pFastBacHTa HBV S 1/S2-TBD. This plasmid was used to produce recombinantbaculovirus (described in example 1), which expressed the chimericantigen-TBD fusion protein: 6×His tag-rTEV protease cleavage site-HBVS1/S2-TBD (See FIGS. 7-9).

Example 3 Construction of HBV Surface Antigen S1/S2/S and HBVS1/S2/S-TBD Fusion Protein Expression Vectors

The DNA encoding the HBV sAg fragment S 1/S2/S was generated from theplasmid pAlt HBV 991 (University of Alberta) template using PCRmethodology. The 5′ primer used for the PCR was (sense) 5′GATAAGGATCCTATGGGAGGTTGGTCATCAAAAC (SEQ ID NO: 7), containing therestriction enzyme Bam HI site. The PCR primer for 3′ terminus was(antisense) 5′ GTCATACTGCGGCCGCGAAATGTATACCCAGAGACAAAAG (SEQ ID NO: 8),containing the restriction enzyme Not I site. Amplified DNA was digestedwith the respective enzymes and ligated with pFastBac HTa expressionvector to generate either the expression plasmid for HBV S1/S2/S or theexpression plasmid pFastBac HTa HBV S1/S2/S-TBD for the fusion protein(see FIGS. 10-11).

Example 4 Construction of HBV Core Antigen and HBV Core-TBD FusionProtein Expression Vectors

HBV produces the Core proteins (Core) to encapsidate the replicatinggenome of the virus. There are two forms of the Core one secreted intocirculation, also known as the “e” antigen and the capsid forming Coreprotein. The present invention also relates to the generation ofexpression plasmids to produce the Core protein as well as the Coreantigen-TBD fusion protein, in insect cells. The DNA encoding the HBVCore protein was generated from the plasmid pAlt HBV 991 template usingPCR technique. The 5′ primer used for the PCR was (sense) 5′TGCGCTACCATGGACATTGACCCTTATAAAG (SEQ ID NO: 9), which contains therestriction enzyme Nco I site and the 3′ primer used was (antisense) 5′TGTCATTCTGCGGCCGCGAACATTGAGATTCCCGAGATTGAG (SEQ ID NO: 10), containingthe restriction enzyme Not I site. The PCR-amplified DNA was digestedwith the respective enzymes and ligated with pFastBac HTa expressionvector to generate either the expression plasmid for HBV Core protein orthe expression plasmid pFastBac HTa HBV Core-TBD for the fusion protein(see FIGS. 13-14).

Example 5 Construction of DHBV Surface Antigen Fragment PreS and DHBVPreS-TBD Fusion Protein Expression Vectors

DHBV has served as a powerful animal model in the development ofantiviral therapy for HBV. Pekin ducks, congenitally infected with DHBVhave been used to study the mechanism of replication of the virus andfor the screening of antiviral compounds. The present invention alsodescribes the chimeric DHBV antigen-TBD molecules that could be used astherapeutic vaccines in DHBV-infected ducks, thus providing a viableanimal model for the feasibility studies for HBV therapeutic vaccines.

The DNA encoding DHBV PreS antigen was produced by PCR from a plasmidpFastBac Hta-DHBV PreS/S (University of Alberta). The 5′ primer used forthe PCR was (sense) 5′ TATTCCGGATTATTCATACCG (SEQ ID NO: 11). The uniquerestriction enzyme site EcoRI, resident on the parent plasmid was usedfor directional cloning. The 3′ primer used was (antisense) 5′TGTCATTCTGCGGCCGCGTTTTCTTCTTCAAGGGGGGAGT (SEQ ID NO: 12), containing therestriction enzyme Not I site. Following PCR amplification, the fragmentwas digested with the restriction enzymes EcoRI and Not I and the DNAfragment was purified on a 1% agarose gel. The fragment was ligated withthe expression plasmid pFastBac HTa at the respective sites to producepFastBac HTa DHBV PreS, which expressed the PreS antigen. The samefragment was also used to ligate with pFastBac HTa-TBD to generate theexpression plasmid pFastBac HTa DHBV PreS-TBD. The production ofbaculovirus stocks from these plasmids and the expression of the PreSand PreS-TBD in High Five™ insect cells were done as described inexample 1.

Example 6 Construction of DHBV Surface Antigen Fragment PreS/S and DHBVPreS/S-TBD Fusion Protein Expression Vectors

DHBV PreS/S DNA was produced by PCR methods using 5′ primer (sense) 5′TATTCCGGATTATTCATACCG (SEQ ID NO: 11) and the 3′ primer (antisense) 5′TGTCATTCAGCGGCCGCGAACTCTTGTAAAAAAGAGCAGA (SEQ ID NO: 13), containingrestriction enzyme Not I site. The unique restriction enzyme site EcoRI,resident on the parent plasmid pFastBac HTa PreS/S (University ofAlberta) was used for directional cloning. This plasmid also was thetemplate for generating the required DNA by PCR. All other protocols forthe production of either the DHBV PreS/S or the fusion proteinPreS/S-TBD are the same as described in the example 5 above.

Example 7 Construction of DHBV Core Antigen and DHBV Core-TBD FusionProtein Expression Vectors

The DNA coding for DHBV Core was generated from pRSet B DHBV Core by PCRusing the following primers. The 5′ terminus primer used was (sense) 5′TGCGCTACCATGGATATCAATGCTTCTAGAGCC (SEQ ID NO: 14), containing therestriction enzyme Nco I site. The 3′ terminus primer used was(antisense) 5′ TGTCATTCTGCGGCCGCGATTTCCTAGGCGAGGGAGATCTATG (SEQ ID NO:15), containing the restriction enzyme Not I site. All other protocolsfor the production of either the DHBV Core or the fusion protein DHBVCore-TBD are the same as described in the example 5 above.

Example 8 Chemically Cross-Linked HBV sAg-Fc (Murine)

HBV sAg was cross linked using the bifunctional cross linking agentdimethyl suberimidate (DMS), a homobifunctional imidoester that reactswith amino groups on the proteins. The unreacted components were removedby gel filtration. The conjugate was characterized with respect to thestoichiometry of sAg/Fc in the conjugate and the fraction containingsAg:Fc at 1:1 ratio was chosen for antigen presentation assays usinghuman monocyte-derived immature Dendritic cells (DCs). Immature DCs werecultured for four days with GM-CSF/IL4, incubated with the sAg-Fcconjugate and matured in the presence of TNFα/IFNα. Autologous CD3+ Tcells were added to the mature DCs. Following three rounds of exposureto the mature DCs, T cell stimulation was quantitated by measuring theproduction of intracellular interferon-γ, using flow cytometry.

Materials:

HBV sAg (US Biologicals; Cat#H 1910-27)

Mouse Polyclonal IgG Fc fragment (Harlan Sera-Lab Ltd., Cat#PP-19-01)DMS (Dimethyl suberimidate. 2HCl) (Pierce Cat #20700)

Cross-linking Buffer 0.1M HEPES pH 8.7 Stop Buffer 0.1 M Tris HCl pH 7.8Elution Buffer Phosphate Buffered Saline (PBS) pH 8.3 Sephadex G 75(Pharmacia)

Methods: Solutions of sAg (100 μg) and Mouse Fc fragment (100 μg), weredialyzed against the cross linking buffer overnight at 4° C. The proteinsolutions were mixed together, DMS reagent was added immediately to afinal concentration of 10 mM, and the mixture was incubated at roomtemperature for 1 hr. The reaction was stopped by the addition of 0.1 MTris HCl pH 7.8. The reaction mixture was loaded on a Sephadex G 75column (0.7×12 cm), and fractions were eluted using elution buffer. 0.5ml fractions were collected and the fractions containing sAg/Fc at amolar ratio of 1:1, as estimated by ELISA using the respectiveantibodies were pooled and used for Antigen Presentation Assays.(Berlyn, et al., supra (2001)).

Results: The levels of intracellular interferon-γ produced in T cells inthe presence of conjugate was substantially higher than the sAg or theFc fragment alone.

Example 9 Chimeric Antigens of Hepatitis C Virus (HCV)

HCV Core-TBD was cloned using the pFastBac HTa vector and thebaculovirus system and expressed in Sf9 and High Five™ insect cells,similar to the HBV fusion proteins. This was done as follows. The DNAencoding the HCV Core fragment was generated from the plasmid pCV-H77c(NIH) template using PCR methodology.

The primers used were: (sense) 5′ CGGAATTCATGAGCACGAATCCTAAAC (SEQ IDNO: 16) containing the restriction enzyme site EcoRI and the 3′ primer(antisense) 5′ GGACTAGTCCGGCTGAAGCGGGCACAGTCAGGCAAGAG (SEQ ID NO: 17)containing the restriction enzyme site Spe I. Amplified DNA was digestedwith EcoRI/Spe I and the fragment was ligated into the plasmid pFastBacHTa TBD (described in example 1) following the digestion with therespective enzymes. This produced the expression plasmid pFastBac HTaHCV Core-TBD. This plasmid was used to produce recombinant baculovirus(described in example 1), which expressed the chimeric antigen (HCVCore-TBD) fusion protein 6×His tag-rTEV protease cleavage site-HCVCore-TBD.

HCV Core Protein was cloned as follows. Amplified DNA was digested withEcoRI/Spe I and ligated with plasmid pFastBac HTa expression vector togenerate the expression plasmid for HCV Core protein. This protein isexpressed with N-terminal 6×His tag and rTEV protease cleavage site.

The following HCV antigens and their respective chimeric antigens(antigen-TBD) have been cloned and are ready for expression.

E1 & E1-TBD:

E2 & E2-TBD

E1 E2 & E1 E2-TBD

NS5A & NS5A-TBD

Example 10 Cloning, Expression and Purification of Recombinant ProteinsUsing a Baculovirus Expression System

Bac-to-Bac™ Baculovirus Expresssion System is commercially availablefrom Invitrogen and the procedures used were as described in the companyprotocols. The gene of interest was cloned into pFastBac HTa donorplasmid and the production of recombinant proteins was based upon theBac-to-Bac™ baculovirus expression system (Invitrogen).

In the next step, the pFastBac HTa donor plasmid containing the gene ofinterest was used in a site-specific transposition in order to transferthe cloned gene into a baculovirus shuttle vector (bacmid). This wasaccomplished in E. coli strain DH10Bac™. The DH10Bac™ cells contain thebacmid, which conferred kanamycin resistance and a helper plasmid, whichencoded the transposase and conferred resistance to tetracycline. Therecombinant pFastBac HTa plasmids with the gene of interest weretransformed into DH10Bac™ cells for the transposition to generaterecombinant bacmids. A 100 μl aliquot of competent DH10Bac™ cells wasthawed on ice, the pFastBac HTa based plasmids were added and themixture was incubated on ice for 30 minutes. The mixture was given aheat shock for 45 seconds at 42° C. and then chilled on ice for 2minutes. The mixture was then added to 900 μL of LB media and incubatedfor 4 hours at 37° C. The transformed cells were serially diluted withLB to 10⁻¹ and 10⁻² and 100 μl of each dilution was plated on Luriabroth (LB) agar plates (supplemented with 50 μg/ml kanamycin, 7 μg/mlgentamicin, 10 μg/ml tetracycline, 100 μg/ml X-gal, and 40 μg/ml IPTG)and incubated for at least 36 hours at 37° C. The gentamicin resistancewas conferred by the pFastBac HTa and the X-gal and IPTG were used todifferentiate between white colonies (recombinant bacmids) from bluecolonies (non recombinant). The white colonies were picked andinoculated into 2 ml of LB (supplemented with 50 μg/ml kanamycin, 7μg/ml gentamicin and 10 μg/ml tetracycline) and incubated overnight at37° C., with shaking. A sterile loop was used to sample a small amountof the overnight culture and the sample was streaked onto a fresh LBagar plate (supplemented with 50 μg/ml kanamycin, 7 μg/ml gentamicin, 10μg/ml tetracycline, 100 μg/ml X-gal, and 40 μg/ml IPTG) and incubatedfor at least 36 hours at 37° C. to confirm a white phenotype.

Recombinant bacmids were isolated by standard protocols (Sambrook,supra); the DNA sample was dissolved in 40 μl of TE (10 mM Tris-HCl pH8, 1 mM EDTA) and used for transfections.

In order to produce baculoviruses, the bacmid was transfected into Sf9insect cells. Sf9 cells (9×10⁵) were seeded into each well of a 6-wellcell culture dish (35 mm wells) in 2 ml of SFM 900 II and allowed toattach for at least 1 hour at 27° C. Transfections were carried outusing Cellfectin® Reagent (Invitrogen, Cat. No. 10362-010) as per theprotocols provided by the supplier of the Sf9 cells. Followingtransfection, the cells were incubated at 27° C. for 72 hours. Themedium containing baculovirus was collected and stored at 4° C. in thedark.

The efficiency of the transfection was verified by checking forproduction of baculoviral DNA. The isolated baculovirus DNA is subjectedto PCR to screen for the inserted gene of interest. The primers used arepFastBac HTa 5′ (sense) TATTCCGGATTATTCATACCG (SEQ ID NO: 3) andpFastBac HTa 3′ (antisense) 5′ CTCTACAAATGTGGTATGGC (SEQ ID NO: 4).Amplified products were separated on an agarose gel (0.8%). Theexpression of the heterologous protein in the cells was verified by SDSpolyacrylamide gel electrophoresis (SDS-PAGE) and Western blots usingthe 6×His tag monoclonal antibody (Clontech) as the probe.

Once production of baculovirus and the expression of protein were beenconfirmed, the virus stock was amplified to produce a concentrated stockof the baculovirus that carry the gene of interest. It is standardpractice in the art to amplify the baculovirus at least two times, andin all protocols described herein this standard practice was adhered to.After the second round of amplification, the concentration of thegenerated baculovirus was quantified using a plaque assay according tothe protocols described by the manufacturer of the kit (Invitrogen). Themost appropriate concentration of the virus to infect insect cells andthe optimum time point for the production of the desired protein wasalso established.

Example 11 Expression of the Recombinant Proteins

Recombinant baculovirus of standardized multiplicity of infection (MOI)were used to infect High Five™ insect cells. For suspension cultures,cells were seeded at a density of 3×10⁵ cells/mL and incubated at 27.5°C. with shaking at 138 rpm until the cell density reached 2-3×10⁶cells/mL. Standardized amounts of the respective recombinant baculoviruswas added to the cells. The incubation temperature was 27.5° C. and theappropriate infection period was standardized for individual proteinexpression. The cells were harvested by centrifugation at 2,500 rpm for10 minutes at 4° C. and used for the purification of the recombinantproteins. Unused portions of cells were snap frozen in liquid nitrogenand stored at −70° C.

Example 12 Purification of Proteins

For purification under denaturing conditions, the cells were lysed in abuffer containing 6 M guanidinium-HCl in 100 mM NaH₂PO₄, 10 mM Tris, 300mM NaCl, 10 mM Imidazole, pH 8.0 (lysis buffer). The suspension wassonicated on ice with 5 pulses of 1 minute per pulse at a power settingof 60 watts, and was mixed at room temperature for 1 hour. The lysatewas centrifuged at 27,000×g for 30 min to remove unbroken cells and celldebris. The supernatant was loaded on to a Ni-NTA agarose (Qiagen) beadcolumn (1×5 cm/100 mL cell lysate), pre-equilibrated with lysis buffer.Following loading, the column was washed with 20 column volumes of 6 Mguanidinium-HCl in 100 mM NaH₂PO₄, 10 mM Tris, 300 mM NaCl, 40 mMImidazole, pH 8.0 (wash buffer 1), followed by washes with 20 columnvolumes of 8 M urea in 100 mM NaH₂PO₄, 10 mM Tris, 300 mM NaCl, 40 mMimidazole, pH 8.0 (wash buffer 2). The bound protein was eluted with abuffer containing 8 M urea, 100 mM NaH₂PO₄, 10 mM Tris, 300 mM NaCl, 250mM imidazole, pH 8 (Elution Buffer). The fractions containing theprotein was pooled and dialyzed against PBS, (Overnight, 4° C.).

Examples 13-16 Use of Chimeric Antigens to Enhance Antigen Presentationby Human PBMC-Derived Dendritic Cells and to Elicit an Immune Responsein T Lymphocytes Example 13 Human PBMC Monocyte Isolation andDifferentiation to DCs

Peripheral blood mononuclear cells (PBMC) were obtained fromFicoll/Histopaque (Sigma) treatment of a leukapheresis cell preparation(Berlyn, et al., supra (2001)). Monocytes were separated from the PBMCpopulation by negative selection using a monocyte isolation kit (Dynal)following the manufacturer's directions. The monocytes were greater than95% pure as assessed by antibody analysis and flow cytometry (CD3⁻,CD19⁻, CD16⁻, CD11a⁺, CD14⁺). Monocytes were washed twice with AIM V(Invitrogen) media containing L-glutamine, streptomycin sulfate (50μg/mL) and gentamicin sulfate (10 μg/mL) with 1% donor matched sera(isolated as described in Berlyn, et al., supra (2001)). Next, themonocytes were cultured in AIM V media containing 2.5% donor matchedsera and the cytokines GM-CSF and IL-4 to differentiate the cells towardthe dendritic cell (DC) lineage. The cells were incubated in 12-welltissue culture plates at 37° C. under a 7% CO₂ atmosphere. The DCs wereused for APAs and ligand binding and uptake studies.

The monocyte-derived DCs (mDC) were harvested on days 1 through 4. Thecells were subsequently washed once with AIM V media with 0.1% BSA(Sigma), and twice with Dulbecco's phosphate buffered saline(Invitrogen) with 0.1% (w/v) BSA (PBSB). The mDC were used in 4° C.labeling or binding assays or in 37° C. binding/uptake assays.

Example 14 Human Dendritic Cell T Cell Stimulation Assay

Antigen presentation assays were performed using human PBMC-deriveddendritic cells according to established protocols (Berlyn, et al.,supra (2001)). Monocytes were generated from leukapheresis samples fromhealthy donors and were depleted of lymphocytes and granulocytes byincubation with anti-CD2, CD7, CD16, CD19, and CD56 antibodies. This wasfollowed by incubation with magnetic bead conjugated anti-mouse IgG andseparation on a magnet (Dynal). Negatively selected cells were greaterthan 95% pure monocytes as characterized by flow cytometry using a broadCD marker panel. Next, monocytes were incubated with IL-4 and GM-CSF(R&D Systems) for 4 days in AIM V plus 2.5% matched human serum togenerate immature dendritic cells. Again, an aliquot of the cells wasstained with a broad CD marker panel to ensure purity and identity ofthe cells. The cells then were loaded with various antigens for 2-4hours at 37° C., and matured with interferon-α and TNF-α for 3 days.Dendritic cells were checked again using flow cytometry for an array ofCD markers to ensure that cells had undergone proper maturation. Theresulting mature, loaded dendritic cells were used for the T cellstimulation assay. A protocol summary for the T cell stimulation assayis presented in schematic form.

T cells were generated from the same monocytes as the dendritic cells bymeans of negative selection using a magnetic T cell isolation kit(Dynal) according to the manufacturer's directions. Mature, loadeddendritic cells (DC-1) were washed thoroughly and added to the T cells(Day 0). The T cells and dendritic cells were incubated for 7 days

On Day 7, the T cells were re-stimulated with matured, loaded dendriticcells (DC-2). An aliquot of the cells was taken 2 hours later (the Day 7aliquot). The Day 7 aliquot was incubated with Brefeldin A (GolgiPlug™,R&D Systems) for 18 hours. The cells of the Day 7 aliquot were thenassayed for intracellular cytokine staining as described below.

The remaining cells were incubated for another 7 days. On Day 14, theremaining cells were stimulated with another batch of mature, loadeddendritic cells (DC-3). An aliquot of the cells was taken 2 hours later(the Day 14 aliquot). The Day 14 aliquot was incubated with Brefeldin A(GolgiPlug™, R&D Systems) for 18 hours. The cells of the Day 14 aliquotwere then assayed for intracellular cytokine staining as describedbelow.

After removal of the D14 aliquot, the remaining cells were incubated forthree days and the supernatant was used for measuring the level ofsecreted interferon-γ by ELISA (Opt E1A ELISA kit, BD Biosciences).

For intracellular cytokine staining, cells were stained withanti-CD3-FITC and anti-CD8-Cy-Chrome for 30 minutes, washed, fixed,permeabilized, and then stained with anti-interferon-γ-PE for 30 minuteson ice. The cells were washed and analyzed by flow cytometry (FACScan,BD Biosciences).

Example 15 Expression of Fc-γ Receptors and CD206 on Maturing DC

There are several receptors on the APCs that bind and take up antigens.The abundance of these receptors on maturing dendritic cells wasevaluated using fluorescent labeled receptor-specific antibodies. FACSanalysis was used to estimate percentage of specific receptor positivecells in the total population of dendritic cells. The degree of receptorexpression was assessed by determination of the relative meanfluorescent intensity and as a function of relative fluorescentintensity (FIG. 30). The expression of CD64 decreased with time inculture and at day 4 was almost negligible. In contrast, CD32, and to alesser extent CD16, continued to be expressed after 4 days of DCculture. On day 0 of culture, there was essentially no CD206 expression,but expression was induced upon culture with IL-4 and GM-CSF, and by day4 CD206 was expressed at very high levels. Thus at day 4, when antigenwas loaded in the antigen presentation assays, the DCs possessed atleast two potential receptors for the binding of chimeric antigens: CD32and CD206. In addition, as shown in FIG. 31, they had the fullcomplement of the co-stimulatory molecules. The expression of HLA-DR(Class II) and HLA-ABC (Class I) also increased with time in culture.Co-stimulatory molecules CD86 (B7.2) and CD80 (B7.1) were expressedthroughout the period of the assay (FIG. 31). These results indicatethat the monocyte-derived DCs were differentiating towards mature DCsand were capable of antigen processing and presentation to T cells. Thecells were used to evaluate the binding and uptake of the chimericantigens in comparison to relevant antibodies.

Example 16 Phenotypic Analysis, Binding and Uptake Assay

For the phenotypic analysis and binding assay, all procedures usingincubations were performed at 4° C.; buffer solutions were also held at4° C. The binding of antigens, chimeric antigens or antibodies wasdetermined by incubating the cells with various concentrations of theagents for 60 minutes in Dulbecco's phosphate buffered saline with 0.1%(u/v) BSA (PBSB).

For phenotypic analysis, cells were incubated with the variousconjugated mAbs at the concentrations recommended by the manufacturerfor 20 minutes. Incubations were performed with 1×10⁵ cells/well in96-well v-bottom plates in a volume of 25 μL/well. Subsequently, thecells were washed twice with PBSB.

For binding analysis, the cells were treated with F(ab′)₂ goatanti-mouse Alexa-488 (10 μg/mL) in PBSB for 20 minutes. The cells werewashed twice with PBSB and either resuspended in PBSB with 2% PF andacquired by FACS or in PBSB and incubated with PE-conjugated CD32 orCD206 specific mAb for 20 minutes before washing twice with PBSB.

To determine the extent of uptake of chimeric antigens (e.g. HBVS1/S2-TBD) compared with IgG1 and IgG2a, cells were incubated withvarious concentrations of the antigen, IgG1 (2C12, the parent mAb fromwhich TBD was produced) or IgG2a (G155-178) for 1 hour at 37° C. in AIMV media with 0.1% BSA. Cells were washed twice in PBSB and fixed withPBS with 2% PF overnight at 4° C. Subsequently, the cells were washedtwice in PBSB and permeabilized with PBS containing 0.1% (w/v) saponin(Sigma) for 40 minutes at 20° C.

The cells were washed twice with PBSB and incubated with F(ab′)₂ goatanti-mouse Alexa-488 (10 μg/mL) in PBSB with 0.1% (w/v) saponin for 20minutes at 4° C. After washing twice in PBSB, the cells were resuspendedin PBSB. A variant of this assay involved treating the cells as abovewith chimeric antigen, IgG1, or IgG2a for 10 minutes followed by theaddition of F(ab′)₂ goat anti-mouse Alexa-488 (10 μg/mL) for 50 minutes.Subsequently the cells were washed and resuspended in PBS with 2% PF.This procedure relied on the ability of the anti-mouse Alexa-488 Ab todirectly bind the S1/S2-TBD, IgG1 or IgG2a molecules.

Cells were acquired by a Becton Dickinson (BD) FACScan fitted withCellquest acquisition and analysis software (BD). A gate was made on theviable cell population as determined by the FSC and SSC scatter profileand ≧10,000 events were acquired. To determine the percentage ofpositive cells, a gate was set based on negative control treated cells(isotype control labeled or cells labeled with F(ab′)₂ goat anti-mouseAlexa-488 alone).

The percent of specific positive cells was calculated as:

$\frac{{\% \mspace{14mu} {positive}\mspace{14mu} {cells}\mspace{14mu} {test}\mspace{14mu} {sample}} - {\% \mspace{14mu} {positive}\mspace{14mu} {cells}\mspace{14mu} {control}}}{100 - {\% \mspace{14mu} {positive}\mspace{14mu} {cells}\mspace{14mu} {of}\mspace{14mu} {control}}} \times 100$

The relative mean fluorescent intensity (MFI) was determined as the MFIof the test sample minus the MFI of the control sample.

Example 17 Construction of pFastBac HTa-TBD, TBD Protein ExpressionVector

The mouse IgG 1 DNA sequences encoding amino acids ofC_(H)1-Hinge-C_(H)2-C_(H)3 region was generated from mRNA isolated fromthe hybridoma (2C12), which produces mAb against HBV surface antigen(sAg). Total mRNA was isolated using Trizol® reagent (Gibco BRL cat. No.15596-026) and the cDNA of the TBD was generated by RT-PCR usingSuperscript First-strand Synthesis (Invitrogen Cat. No. 11904-018). ThePCR primers contained linker sequences encoding the linker peptide—SRPQGGGS— (SEQ ID NO: 28) at the 5′ terminus, a unique Not I site atthe 5′-end and a unique Hind III restriction site at the 3′ end. Theresulting cDNA contains (5′ Not I)-linker sequence-C_(H)1(VDKKI)-C_(H)2-C_(H)3-(3′ Hind III). Following digestion with therespective enzymes, the fragment is ligated with pFastBac HTa expressionvector plasmid (Invitrogen) using the same restriction enzyme sites togenerate pFastBac HTa-TBD. The 5′ primer used for PCR amplification was(Sense) 5′ TGTCATTCTGCGGCCGCAAGGCGGCGGATCCGTGGACAAGAAAATTGTG CCCAGG (SEQID NO: 1) and the 3′ primer was (antisense) 5′ACGAATCAAGCTTTGCAGCCCAGGAGAGTGGGAGAG (SEQ ID NO: 2), which contained theNot I and Hind III sites, respectively. The following is the protocolused for directional cloning. The generated fragment was digested withthe respective enzymes, purified on agarose gel and cloned into thevector plasmid. The DNA sequence and the correctness of the ORF wereverified by standard sequencing methods. Nucleotide sequence of the ORFof TBD in the plasmid pFastBac HTa-TBD and the deduced amino acidsequences of the expressed TBD protein from the ORF are shown in FIG. 6.

Example 18 Expression and Purification of TBD Protein

Recombinant baculovirus of standardized multiplicity of infection (MOI)were used to infect High Five™ insect cells. For suspension cultures,cells were seeded at a density of 3×10⁵ cells/mL and incubated at 27.5°C. with shaking at 138 rpm until the cell density reached 2−3×10⁶cells/mL. Recombinant baculovirus was added to the cells. For theexpression of TBD the MOI used was 10 pfu/cell. The incubation at 27.5°C. was continued for 48 hrs. The cells were harvested by centrifugationat 2,500 rpm for 10 minutes at 4° C. and used for the purification ofthe recombinant proteins.

TBD protein was expressed in Express Five Insect cells, purified asdescribed in Example 12. The protein was subjected to electrophoresis ona 12% polyacrylamide gel and the coomassie blue-stained band is shown.

Example 19 Construction of HBV Surface Antigen S1/S2 and HBV S1/S2-TBDChimeric Fusion Protein Plasmids

The DNA encoding the HBV sAg fragment S1/S2 was generated from theplasmid pRSetB HBV S1/S2 template using PCR methodology. The primersused were: (sense) 5′ GGATCTGTACGACGATGACG (SEQ ID NO: 5) and the 3′primer (antisense) 5′ AGTCATTCTGCGGCCGCGAGTTCGTCACAGGGTCCCCGG (SEQ IDNO: 6) containing the restriction enzyme site Not I. The 5′ endcontained a unique Bam HI site derived from the parent plasmid that wasused for ligations. Amplified DNA was digested with Bam HI/Not I andligated with pFastBac HTa expression vector to generate the expressionplasmid for HBV S1/S2 protein. The fragment was ligated with the plasmidpFastBac HTa-TBD (described in Example 1) following the digestion withthe respective enzymes. This produced the expression plasmid pFastBacHTa HBV S1/S2-TBD. This plasmid was used to produce recombinantbaculovirus (as described in Example 1), which expressed the chimericantigen-TBD fusion protein: 6×His tag-rTEV protease cleavage site-HBVS1/S2-TBD. Nucleotide and deduced amino acid sequences from the ORFs ofplasmid pFastBac HTa HBV S1/S2 are shown in FIG. 9. Nucleotide anddeduced amino acid sequences from the ORFs of plasmid pFastBac HTa HBVS1/S2-TBD are shown in FIG. 8.

Example 20 Expression and Purification of HBV Surface Antigen S1/S2 andHBV S1/S2-TBD Chimeric Fusion Proteins

Recombinant bacmids of standardized multiplicity of infection (MOI) wereused to infect High Five™ insect cells. For suspension cultures, cellswere seeded at a density of 3×10⁵ cells/mL and incubated at 27.5° C.with shaking at 138 rpm until the cell density reached 2−3×10⁶ cells/mL.Recombinant baculovirus was added to the cells. For the expression ofthe fusion protein HBV S 1/S2-TBD, the MOI was 1 pfu/cell and for S1/S2,2 pfu/cell was used. The incubation at 27.5° C. was continued for 48hrs. The cells were harvested by centrifugation at 2,500 rpm for 10minutes at 4° C. and used for the purification of the recombinantproteins.

Expression of S1/S2-TBD was performed in High Five™ cells (Trichoplusiani BTI-Tn-5B1-4) grown in Express Five SFM media. The High Five™ cellswere grown in a shaker culture at 27.5° C. until the cell densityreached 2.5×10⁶ cells/ml. Usually, a 250 ml culture is prepared. Theculture was infected with HBV S1/S2-TBD baculovirus at a multiplicity ofinfection (MOI) of 1 pfu/cell and incubated at 27.5° C. for 48 hrs withshaking. Infected cells were harvested by centrifugation at 4000×g on aJA-10 (Beckman) rotor for 10 minutes. The cells were stored at −70° C.until purification were performed.

For purification, 40 ml of ice-cold lysis buffer (6M guanidinehydrochloride, 0.1 M NaH₂PO₄, 10 mM Tris, 500 mM NaCl, 10 mM imidazole,pH 8.0) was added to a frozen cell pellet. The cells were sonicated onice for 5 pulses at 1 minute per pulse at 78-81 W and stirred at roomtemperature for 1 hour. The lysate was clarified by centrifugation at27000×g on a JA-25.50 rotor (Beckman) for 30 min. Purification wasperformed on Ni-NTA Superflow. A 1.5×12 cm column was packed with 3 mlof Ni-NTA Superflow and equilibrated with 10 column volumes of lysisbuffer. The clarified lysate was loaded onto the column. First, thecolumn was washed with lysis buffer until the OD₂₈₀ was <0.01. Next, thecolumn was washed with 6M guanidine hydrochloride, 0.1 M NaH₂PO₄, 10 mMTris, 500 mM NaCl, 40 mM imidazole, pH 8.0 until the OD₂₈₀ is <0.01.Then the column was washed with 8 M urea, 0.1 M NaH₂PO₄, 10 mM Tris, 500mM NaCl, 40 mM imidazole, pH 8.0 until the OD₂₈₀ was <0.01. Elution wasperformed with 8M urea, 0.1 M NaH₂PO₄, 10 mM Tris, 500 mM NaCl, 250 mMimidazole, pH 8.0 and 0.5 ml fractions were collected. The fractionswere analyzed by OD₂₈₀ for protein. HBV S1/S2 and TBD protein fractionswere dialyzed against 10 mM NaH₂PO₄, 0.3 M NaCl, pH 8.0.

S1/S2-TBD was dialyzed against 8M urea, 0.1 M NaH₂PO₄, 10 mM Tris, pH8.0 with 3 changes and was subjected to further purification as follows.A 1 ml bed of DEAE Sepharose Fast Flow was equilibrated with 8M urea,0.1 M NaH₂PO₄, 10 mM Tris, pH 8.0. The dialyzed S1/S2-TBD was added tothe DEAE Sepharose Fast Flow and mixed together for 2 hours at roomtemperature. The mixture was centrifuged at 2500 rpm for 2 mM and thesupernatant was collected.

Purified S1/S2-TBD was subjected to refolding. The DEAE purifiedS1/S2-TBD was reduced by adding 10 mM DTT and incubated for 30 minutesat room temperature. The reduced S1/S2-TBD was dialyzed against 4 Murea, 0.1 M NaH₂PO₄, 10 mM Tris, 150 mM NaCl, pH 8.0 at 4° C. for atleast 6 hrs. The buffer was changed to 2M urea, 0.1 M NaH₂PO₄, 10 mMTris, 150 mM NaCl, pH 8.0 and dialysis was continued at 4° C. After atleast 6 hrs, the dialysis buffer was changed to 1 M urea, 0.1 M NaH₂PO₄,10 mM Tris, 150 mM NaCl, 200 mM L-arginine, 0.5 mM oxidized glutathione(GSSG), pH 8.0 and dialysis was continued at 4° C. overnight. Followingthis, the buffer was changed to 0.5 M urea, 0.1 M NaH₂PO₄, 10 mM Tris,150 mM NaCl, 200 mM L-arginine, 0.5 mM GSSG, pH 8.0 and dialysis wascontinued at 4° C. overnight. Finally, the sample was dialyzed against10 mM NaH₂PO₄, 150 mM NaCl, pH 8.0 at 4° C. for at least 6 hrs. The laststep was repeated 2 more times.

Example 21 Binding of Chimeric Antigens to Maturing DCs

The chimeric antigen S1/S2-TBD binds to maturing DCs with highefficiency (FIG. 32). The extent of binding of S1/S2-TBD relative tomurine IgG1 and IgG2a to maturating DC was compared. DCs were isolatedat various days of ex vivo culture (from day 0 to day 4) and treatedwith S1/S2-TBD (10 μg/mL) or with murine IgG1 (clone 2C12) or IgG2a(clone G155-178, 90 μg/mL) for 1 hour at 4° C. Subsequently, binding wasdetected with a F(ab′)₂ anti-mouse IgG conjugated to Alexa 488 asdescribed in Example 16. The binding of S1/S2-TBD relative to IgG1 andIgG2a on DC after 1 and 4 days of culture is shown in FIGS. 33 and 34.S1/S2-TBD binding was clearly much greater than the binding of eitherIgG1 or IgG2a with more S1/S2-TBD binding evident on day 1 than on day4. These experiments clearly demonstrated that S1/S2-TBD was bound withhigh efficiency to the maturing DC.

Example 22 A High Proportion of Maturing DCs Bind Chimeric AntigenS1/S2-TBD

A large proportion of maturing DCs bind S1/S2-TBD. The binding ofS1/S2-TBD in comparison to murine IgG2a and IgG1 was measured as afunction of phenotypic changes on day 2 of the maturation of DCs asdescribed in Example 16. DCs were isolated at various days of culture(from day 0 to day 4) and were treated with S1/S2-TBD (10 μg/mL), murineIgG1 (clone 2C12), or IgG2a (clone G155-178, 90 μg/rap for 1 hour at 4°C. Subsequently, binding was detected with a F(ab)₂ anti-mouse IgGconjugated to Alexa 488. The binding of S1/S2-TBD relative to IgG1 andIgG2a on DC after 1 and 4 days of culture is shown in FIGS. 33 and 34.S1/S2-TBD binding was clearly much greater than the binding of eitherIgG1 or IgG2a with more S1/S2-TBD binding evident on day 1 than day 4.Thus, these experiments demonstrated that a large proportion of maturingDCs bind S1/S2-TBD The proportion of DCs that bind S1/S2-TBD was muchgreater than either IgG2a or IgG1. Furthermore, the degree of binding ofS1/S2-TBD was several orders of magnitude greater than that of theimmunoglobulins.

The chimeric Antigen S1/S2-TBD binds to DCs more efficiently than IgG1or IgG2a on days 1 and 4 of culture.

Example 23 Chimeric Antigen S1/S2-TBD is Taken up by DCs with HighEfficiency

The uptake of S1/S2-TBD in comparison to murine IgG1 and IgG2a wasestimated as a function of concentration on day 4 of DC maturation. Theuptake was quantified at 37° C. for 1 hour and the results are shown inFIG. 35.

There was a linear increase in the uptake of S1/S2-TBD withconcentration. IgG 1 was taken up at a much lower level and there wasvery little uptake of IgG2a. Therefore, the chimeric antigen S1/S2-TBDis taken up by the DCs more efficiently than immunoglobulins.

Example 24 Correlation of CD32/CD206 Expression and S1/S2-TBD Binding toMaturing DCs

There is a direct correlation between the expression of CD32/CD206receptors and S1/S2-TBD binding to maturing DCs. Since it was known thatmurine IgG1 binds to human CD32, it was expected that S1/S2-TBD, whichcontains the murine Fc component of IgG1, would also bind CD32.Furthermore, S1/S2-TBD by virtue of its high mannose glycosylation,would also be expected to bind to DC through the CD206 receptor.

The dot plots in FIG. 36 show S1/S2-TBD binding (10 μg/mL) and CD32expression as well as S1/S2-TBD binding and CD206 expression. There wasa direct correlation between the extent of S1/S2-TBD binding and thedegree of CD32 expression, which was relatively heterogeneous, i.e.,there was a broad degree of expression. These results demonstrate thatS1/S2-TBD binds to CD32, and that the greater the expression of CD32,the greater was the degree of binding of the chimeric antigen S1/S2-TBD. The dot plot of S 1/S2-TBD binding and CD206 expression showsthat the vast majority of cells expressing CD206 also bound S 1/S2-TBD Asmall percentage of the cell population was CD206 negative and wasconsequently negative for S1/S2-TBD binding. Therefore both CD32 andCD206 receptors correlate with the binding of S1/S2-TBD.

Example 25 The Binding and Uptake of S1/S2-TBD is Primarily Via CD32with CD206 Involved to a Lesser Extent

The uptake of S1/S2-TBD in comparison to murine IgG1 and IgG2a wasestimated as a function of concentration on day 4 of DC maturation. Theuptake was quantified at 37° C. for 1 hour in the presence and absenceof inhibitors of CD32 and CD206 and the results are shown in FIG. 37.There was a progressive increase in the binding of the chimeric antigenwith its concentration. Incubation of the cells with a highconcentration of mouse Fcγ fragment abolished this binding, whereasmannan, an inhibitor of CD206 receptor binding, had only a marginaleffect. Therefore, CD32 may be the primary receptor involved in thebinding and uptake of the chimeric antigen.

Example 26 Glycosylated HBV S1/S2 Antigen Produced in Insect Cells Bindsto DCs Through CD206 Receptors

The insect cell pathway of protein glycosylation is different from thatof mammalian cells in that proteins synthesized in insect cells undergoglycosylation that results in high mannose content and a lack ofterminal sialic acid residues in the secreted protein (Altman, et al.,Glycoconjug 16:109-123 (1999)).

HBV S1/S2, the antigen component of the chimeric antigen was expressedin both E. coli (no glycosylation) and in High Five™ insect cells (highmannose glycosylation). These antigens were compared for their bindingto DCs. Glycosylated protein showed better binding and uptake by DCs(FIG. 38).

Example 27 Chimeric Antigen S1/S2-TBD Elicited T Cell Responses asMeasured by Interferon-γ Production

The T cell response was greater with S1/S2-TBD treatment than witheither of its two components measured individually. DCs were loaded withS1/S2 antigen, TBD, or S1/S2-TBD and presented to T cells in an antigenpresentation assay as described in example 14. T cell stimulation wasevaluated by measuring intracellular and secreted interferon-γ levels.The results are presented in FIGS. 39 and 40. The chimeric antigen S1/S2-TBD induced the production of higher interferon-γ levels comparedto either the IRD or the TBD domain of the molecule when tested alone,at equivalent concentrations. It should be pointed out that 5 μg dose ofS1/S2-TBD contains roughly 2.5 μg each of the components.

Example 28 Interferon-γ Production Following S1/S2-TBD AntigenPresentation by DCs

Interferon-γ production and secretion by CD3⁺ T cells increased in aconcentration dependent manner following S 1/S2-TBD antigen presentationby DCs. Purified S1/S2-TBD was used in antigen presentation assays usinghuman PBMC-derived DCs, and the secreted and intracellular interferon-γlevels were measured in T cells following three rounds of antigenpresentation. FIG. 41 presents intracellular levels and FIG. 42 showsthe secreted levels. The results are the mean of three estimates.

Various concentrations of S1/S2-TBD were tested for the T cell response.The effect of S1/S2-TBD was greater than the tetanus toxoid treatment atsimilar concentrations. At concentrations lower than 5 μg/mL, thechimeric antigen elicited a concentration dependent increase in theproduction and secretion of interferon-γ. The positive response at lowconcentrations would be beneficial with respect to the dose necessaryfor vaccination and the cost of manufacturing of a vaccine.

Example 29 Glycosylation of HBV S1/S2 Antigen Imparts Immunogenicity tothe Antigen and Generates Higher T Cell Responses

Glycosylation of HBV S1/S2 elicits increased immunogenicity and T Cellresponses. The insect cell pathway of protein glycosylation is differentfrom that of mammalian cells in that proteins synthesized in insectcells undergo glycosylation that results in high mannose content and alack of terminal sialic acid residues in the secreted protein (Altman,et al., supra).

HBV S1/S2, the antigen component of the chimeric antigen was expressedin both E. coli (no glycosylation) and in High Five™ insect cells (highmannose glycosylation). These antigens were compared for T cellresponses when presented by DCs. Both intracellular and secretedinterferon-γ levels were measured and the results are presented in FIGS.43 and 44. HBV S 1/S2 expressed in insect cells generated a higher levelof both intracellular and secreted interferon, as compared to theunglycosylated protein expressed in E-coli.

Example 30 Construction of HBV Core Antigen and HBV Core-TBD FusionProtein Expression Vectors

HBV produces the Core proteins (Core) to encapsidate the replicatinggenome of the virus. There are two forms of the Core; one secreted intocirculation, also known as the “e” antigen; and other is the capsidforming Core protein. The present invention also relates to thegeneration of expression plasmids to produce the Core protein as well asthe Core antigen-TBD fusion protein in insect cells, similar to examplesdescribed in Example 19. The DNA encoding the HBV Core protein wasgenerated from the plasmid pAlt HBV 991 template using PCR technique.The 5′ primer used for the PCR was (sense) 5′TGCGCTACCATGGACATTGACCCTTATAAAG (SEQ ID NO: 9) that contains therestriction enzyme Nco I site and the 3′ primer used was (antisense) 5′TGTCATTCTGCGGCCGCGAACATTGAGATTCCCGAGATTGAG (SEQ ID NO: 10), containingthe restriction enzyme Not I site. The PCR-amplified cDNA was digestedwith the respective enzymes and ligated with pFastBac HTa expressionvector to generate either the expression plasmid for HBV Core protein orthe expression plasmid pFastBac HTa HBV Core-TBD fusion protein.Nucleotide and deduced amino acid sequences from the ORFs of plasmidpFastBac HTa HBV Core are shown in FIG. 15. Nucleotide and deduced aminoacid sequences from the ORFs of plasmid pFastBac HTa HBV Core-TBD areshown in FIG. 14.

Example 31 Construction of DHBV Surface Antigen PreS/S and DHBVPreS/S-TBD Fusion Protein Expression Vectors

DHBV has served as a powerful animal model in the development ofantiviral therapy for HBV. Pekin ducks, congenitally infected with DHBVhave been used to study the mechanism of replication of the virus andfor the screening of antiviral compounds. The present invention alsodescribes the chimeric DHBV antigen-TBD molecules that could be used astherapeutic vaccines in DHBV-infected ducks, thus providing a viableanimal model for the feasibility studies for a HBV therapeutic vaccines.

DNA encoding DHBV PreS/S was produced by PCR methods from templateplasmid pFastBac HTa PreS/S (University of Alberta) using 5′ primer(sense) 5′ TATTCCGGATTATTCATACCG (SEQ ID NO: 11) and the 3′ primer(antisense) 5′ TGTCATTCAGCGGCCGCGAACTCTTGTAAAAAAGAGCAGA (SEQ ID NO: 13),containing restriction enzyme Not I site. The unique restriction enzymesite EcoRI, resident on the parent plasmid pFastBac HTa PreS/S was usedfor directional cloning. All other protocols for the production ofeither the DHBV PreS/S or the fusion protein PreS/S-TBD are the same asdescribed in Example 19. Nucleotide and deduced amino acid sequencesfrom the ORFs of plasmid pFastBac HTa DHBV PreS/S are shown in FIG. 21.Nucleotide and deduced amino acid sequences from the ORFs of plasmidpFastBac HTa DHBV PreS/S-TBD are shown in FIG. 19.

Example 32 Construction of DHBV Core antigen and DHBV Core-TBD FusionProtein Vector Plasmids

The DNA coding for DHBV Core was generated by PCR using the followingprimers. The 5′ terminus primer used was (sense) 5′TGCGCTACCATGGATATCAATGCTTCTAGAGCC (SEQ ID NO: 14), containing therestriction enzyme Nco I site. The 3′ terminus primer used was(antisense) 5′ TGTCATTCTGCGGCCGCGATTTCCTAGGCGAGGGAGATCTATG (SEQ ID NO:15), containing the restriction enzyme Not I site. All other protocolsfor the production of either the DHBV Core or the fusion protein DHBVCore-TBD are the same as described in the example 4 above. Nucleotideand deduced amino acid sequences from the ORFs of plasmid pFastBac HTaDHBV Core are shown in FIG. 24. Nucleotide and deduced amino acidsequences from the ORFs of plasmid pFastBac HTa DHBV Core-TBD are shownin FIG. 23.

Example 33 Construction of pFastBac HTa HCV Core (1-191) Antigen and theChimeric Antigen pFastBac HTa HCV Core (1-191)-TBD Fusion Protein VectorPlasmids

The DNA encoding the HCV Core was generated from the plasmid pCV-H77Ctemplate (University of Alberta) using PCR methodology. The primers usedwere: (sense) 5′ CGGAATTCATGAGCACGAATCCTAAAC (SEQ ID NO: 16) containingthe unique restriction enzyme site EcoRI and the 3′ primer (antisense)5′ GGACTAGTCCGGCTGAAGCGGGCACAGTCAGGCAAGAG (SEQ ID NO: 17) containing theunique restriction enzyme site Spe I. Amplified DNA was digested withEcoRI/Spe I and ligated with pFastBac HTa expression vector digestedwith the same two enzymes. The expression plasmid for HCV Core proteinwas generated with this method. The fragment was ligated with theplasmid pFastBac HTa (described in Example 19) following the digestionwith the respective enzymes. This produced the expression plasmidpFastBac HTa HCV Core. This plasmid was used for the transposition inDH10Bac™ and the recombinant Bacmids used for Sf9 insect celltransfections. The resulting baculovirus carrying the gene of interestwas optimized for MOI and the time for efficient protein expression(described in example 19). The generation of recombinant expressionplasmid pFastBac HTa-HCV Core-TBD was achieved through similarprotocols. The PCR-amplified DNA was digested with EcoRI/Spe I and thepurified fragment was ligated with the plasmid pFastBac HTa-TBD(described in example 19) following the digestion with the respectiveenzymes. This produced the expression plasmid pFastBac HTa HCV Core-TBD.This plasmid was used to produce recombinant baculovirus that expressedthe chimeric antigen-TBD fusion protein: 6×His tag-rTEV proteasecleavage site-HCV Core-TBD. Nucleotide and deduced amino acid sequencesfrom the ORFs of plasmid pFastBac HTa HCV Core (1-191) are shown in FIG.45. Nucleotide and deduced amino acid sequences from the ORFs of plasmidpFastBac HTa HCV Core (1-191)-TBD are shown in FIG. 46. All otherprotocols are described in example 19.

Example 34 Expression and Purification of HCV Core Antigen and HCVCore-TBD Chimeric Fusion Protein

Recombinant bacmids of standardized multiplicity of infection (MOI) wereused to infect High Five™ insect cells. For suspension cultures, cellswere seeded at a density of 3×10⁵ cells/mL and incubated at 27.5° C.with shaking at 138 rpm until the cell density reached 2−3×10⁶ cells/mL.Recombinant baculovirus was added to the cells. For HCV Core, infectionsof High Five™ cells were performed at an MOI of 1 pfu/cell. Cells insuspension were grown to mid-log phase and infected with the recombinantbaculovirus at this MOI. These infected cultures were incubated for 48hours and then the cells were harvested. For HCV Core-TBD, infections ofHigh Five™ cells were done at an MOI of 1 pfu/cell and for 72 hours.

Purification of Proteins: The purification of HCV Core and HCV Core-TBDwas done under denaturing conditions as follows. The cells were lysed ina buffer containing 6 M Guanidinium-HCl, 0.1 M Na₂HPO₄, 0.01 M Tris-HClpH 8.0, 0.01 M Imidazole, (lysis buffer). The suspension was sonicatedon ice with 5 pulses of 1 minute per pulse at a power setting of 60watts, and was mixed at room temperature for 1 hour. The lysate wascentrifuged at 27,000×g for 30 min to remove unbroken cells and celldebris. The supernatant was mixed for 1 hr with Ni-NTA agarose (Qiagen)beads (5 mL/100 mL cell lysate), pre-equilibrated with lysis buffer.Following the mixing step, the beads were loaded on to a column and waswashed with a minimum of 20 column volumes of 8M Urea, 0.1 M Na₂HPO₄,0.01 M Tris-HCl pH 8.0, 0.02M Imidazole (wash buffer), until the OD₂₈₀was <0.01. The bound protein was eluted in a buffer containing 8M Urea,0.1 M Na₂HPO₄, 0.01 M Tris-HCl pH 8, 0.25 M imidazole.

HCV Core-TBD was separated from other proteins by gel filtration. Thepeak elution fractions from Ni-NTA agarose column were loaded on aSephadex G100 (Pharmacia) gel filtration column and the column waseluted with 8M Urea, 0.1 M Na₂HPO₄, 0.01 M Tris-HCl, pH 8.0. Thefractions containing HCV Core-TBD were pooled and dialyzed against PBS(phosphate buffered saline).

HCV Core antigen and the fusion protein HCV Core-TBD fusion protein wereexpressed in High Five™ insect cells, and purified; Coomassieblue-stained HCV Core was run on a 12% polyacrylamide gel. Core-TBD waspurified and a Western blot using 6×His monoclonal antibody.

Example 35 Construction of pFastBac HTa HCV Core (1-177) Antigen andpFastBac HTa HCV Core (1-177)-TBD Fusion Protein Plasmid Vectors

The DNA coding for HCV Core (1-177) was generated by PCR using thefollowing primers. The 5′ terminus primer used was (sense) 5′CGGAATTCATGAGCACGAATCCTAAAC (SEQ ID NO: 18), containing the restrictionenzyme EcoRI site. The 3′ terminus primer used was (antisense) 5′GGACTAGTCCGAAGATAGAGAAAGAGC (SEQ ID NO: 19), containing the restrictionenzyme Spe I site. Following digestion with the two enzymes, the DNAfragment was ligated with plasmid pFastBac HTa to generate pFastBac HTaHCV (Core 1-177) and with pFastBac HTa-TBD to generate the expressionplasmid pFastBac HTa HCV Core (1-177)-TBD. All other protocols for theproduction of either the HCV Core (1-177) antigen or the chimericantigen fusion protein HCV Core (1-177)-TBD are the same as described inexample 19. Nucleotide sequence and the deduced amino acid sequence of6×His-rTEVprotease site-HCV Core (1-177) are shown in FIG. 47.Nucleotide sequence and the deduced amino acid sequence of6×His-rTEVprotease site-HCV Core (1-177)-TBD are shown in FIG. 48.

Example 36 Construction of pFastBac HTa HCV NS5A Antigen and pFastBacHTa HCV NS5A-TBD Fusion Protein Expression Vector Plasmids

The DNA encoding the HCV NS5A fragment was generated from the plasmidpCV-H77C (University of Alberta) template using PCR methodology. The 5′primer used form the PCR was (sense) 5′ CCGGAATTCTCCGGTTCCTGGCTAAGG (SEQID NO: 20) containing the restriction enzyme EcoRI site. The PCR primerfor 3′ terminus was (antisense) 5′ GGACTAGTCCGCACACGACATCTTCCGT (SEQ IDNO: 21) containing the restriction enzyme Spe I site. Amplified DNA wasdigested with the respective enzymes and ligated with pFastBac HTaexpression vector to generate either the expression plasmid for HCV NS5Aor it was ligated with the expression plasmid pFastBac HTa-TBD togenerate the expression plasmid pFastBac HTa HCV NS5A-TBD fusionprotein.

Nucleotide sequence and the deduced amino acid sequence of6×His-rTEVprotease site-HCV NS5A are shown in FIG. 49. Nucleotidesequence and the deduced amino acid sequence of 6×His-rTEVproteasesite-HCV NS5A-TBD are shown in FIG. 50.

Example 37 Construction of pFastBac HTa HCV E1 Antigen and pFastBac HTaHCV E1-TBD Fusion Protein Expression Vectors

Plasmid pFastBac HTa HCV E1 and pFastBac HTa HCV E1-TBD, which are usedto express HCV envelope protein E1 and the respective chimeric antigenE1-TBD fusion protein, were generated as follows. The DNA encoding theE1 protein was generated from the plasmid pCV-H77C template using PCRtechnique. The 5′ primer used for the PCR was (sense) 5′CCGGAATTCTACCAAGTGCGCAATTCCT (SEQ ID NO: 22), which contains therestriction enzyme EcoRI site and the 3′ primer used was (antisense) 5′GGACTAGTCCTTCCGCGTCGACGCCGGCAAAT (SEQ ID NO: 23), containing therestriction enzyme Spe I site. The PCR-amplified cDNA was digested withthe respective enzymes and ligated with pFastBac HTa expression vectorto generate the expression plasmid pFastBac HTa HCV E1 for theexpression of HCV E1 protein. The digested DNA fragment was ligated withpFastBac HTa-TBD to generate the plasmid pFastBac HTa HCV E1-TBD, whichwas used to express HCV E1-TBD fusion protein.

FIG. 51 shows the nucleotide and the deduced amino acid sequences of6×His-rTEVprotease site-HCV E1 in the open reading frame of theexpression plasmid. FIG. 52 shows nucleotide and the deduced amino acidsequences of 6×His-rTEVprotease site-HCV E1-TBD chimeric antigen fusionprotein.

Example 38 Construction of pFastBac HTa HCV E2 Antigen and pFastBac HTaHCV E2-TBD Fusion Protein Expression Vectors

The DNA encoding HCV E2 antigen was produced by PCR from a plasmidpCV-H77C. The 5′ primer used for the PCR was (sense) 5′GCGGAATTCACCCACGTCACCGGGGGAAATGC (SEQ ID NO: 24) containing a uniquerestriction enzyme site EcoRI that is used for directional cloning. The3′ primer used was (antisense) 5′ GGACTAGTCCAGCCGCCTCCGCTTGGGATATGAGT(SEQ ID NO: 25) containing the restriction enzyme Spe I site. FollowingPCR amplification, the fragment was digested with the restrictionenzymes EcoRI and Spe I an the DNA fragment was purified and ligatedwith the expression plasmid pFastBac HTa at the respective sites toproduce pFastBac HTa HCV E2, which expressed the E2 antigen. The samefragment was also used to ligate with pFastBac HTa-TBD to generate theexpression plasmid pFastBac HTa HCV E2-TBD, which expressed the chimericantigen fusion protein HCV E2-TBD. The production of baculovirus stocksfrom these plasmids and the expression of the E2 and E2-TBD in HighFive™ insect cells were done as described in previous examples.

FIG. 53 shows the nucleotide and the deduced amino acid sequences of6×His-rTEVprotease site-HCV E2 in the open reading frame of theexpression plasmid. FIG. 54 shows nucleotide and the deduced amino acidsequences of 6×His-rTEVprotease site-HCV E2-TBD chimeric antigen fusionprotein.

DNA encoding HCV E1/E2 was produced by PCR methods from the plasmidpCV-H77C using 5′ primer (sense) 5′ CCGGAATTCTACCAAGTGCGCAATTCCT (SEQ IDNO: 26) containing the restriction enzyme site EcoRI and the 3′ primer(antisense) 5′ GGACTAGTCCAGCCGCCTCCGCTTGGGATATGAGT (SEQ ID NO: 27)containing the restriction enzyme site Spe I. Restrictionenzyme-digested DNA fragment was cloned into the respective sites ofeither pFastBac HTa to generate pFastBac HTa HCV E1/E2 or pFastBacHTa-TBD to generate pFastBac HTa HCV E1/E2-TBD. All other protocols forthe production of either the E1/E2 antigen or the fusion proteinE1/E2-TBD are the same as described in the example above.

FIG. 55 shows the nucleotide and the deduced amino acid sequences of6×His-rTEVprotease site-HCV E1/E2 in the open reading frame of theexpression plasmid. FIG. 56 shows nucleotide and the deduced amino acidsequences of 6×His-rTEVprotease site-HCV E1/E2-TBD chimeric antigenfusion protein.

Conclusions from Examples 10-38

1. A new class of Chimeric Antigens is designed in order to incorporateantigen and antibody components in the molecule.2. Antigen components can be derived from infectious agents or cancerantigen.3. Antibody components are xenotypic, preferably of murine origin, inthe case of chimeric antigens for administration to humans.4. Chimeric antigen fusion proteins, TBD and the respective antigenshave been produced by recombinant techniques.5. Chimeric antigen fusion proteins, TBD and the respective antigenshave been produced (expressed) in a heterologous expression system(insect cells).6. By virtue of the expression in insect cells, the proteins havemannose glycosylation content.7. Chimeric antigens include fusion proteins from HBV surface antigens(S1/S2), and/or HBV Core and TBD, derived from the murine mAb 2C12.8. Chimeric antigens include fusion proteins of DHBV surface antigensPreS/S, Core and TBD.9. The following antigens from HCV have been cloned and expressed ininsect cell expression systems. HCV Core (1-191), HCV Core (1-177), HCVNS3, HCV NS5A, HCV E1, HCV E2, HCV E1/E2.10. Chimeric antigen fusion proteins of HCV include HCV Core (1-191),HCV Core (1-177), HCV NS3, HCV NS5A, HCV E1, HCV E2, HCV E1/E2 and TBD.11. Chimeric antigen fusion protein HCV Core (1-191)-TBD and HCV Core(1-191) have been expressed and purified.12. Chimeric antigen fusion protein HBV surface antigen S1/S2-TBD andHBV surface antigen S1/S2 have been expressed and purified.13. The fusion proteins bind to and are internalized by antigenpresenting cells (Human PBMC-derived DCs).14. Binding and uptake is via Fey receptors CD32 and possibly throughCD64.15. Binding and uptake can occur via CD206, the mannose macrophagereceptor.16. Mannose glycosylation augments the binding and uptake of theantigens via CD206.17. Chimeric antigen fusion protein HBV surface antigen S1/S2-TBDenhances the antigen presentation by professional antigen presentingcells (DCs).18. DCs loaded with the Chimeric antigen fusion protein HBV surfaceantigen S1/S2-TBD, on presentation to T cells, elicit an immuneresponse.19. The immune response can be measured as an increase in intracellularand secreted interferon-γ.

Example 39 Maturation and Loading of Dendritic Cells

Peripheral blood mononuclear cells (PBMCs) were thawed by the additionof AIM-V (ratio of 9 ml of AIM-V added to 1 ml of frozen cells). Thecells were then centrifuged at 200×g for 5 min, the supernatant removed,and the cells resuspended in AIM-V/1% matched serum and added to eithera 100 mm culture dish or a T-25 culture flask. The PBMCs were incubatedfor 1 hr at 37° C. in a humidified incubator under 7% CO₂. To removenon-adherent cells, the culture was triturated several times, thesupernatant discarded, and the cells washed once with AIM-V medium.Monocytes were harvested with a cell scraper and centrifuged at 300×gfor 5 min. The cell pellet was re-suspended in AIM-V/2.5% matched serumat 2×10⁶ cells/ml and seeded into a 24-well dish. Th IL-4 and GM-CSF(1000 IU/ml each) were added to drive the differentiation of monocytesinto immature DCs. Antigen was added to immature DCs within 4 to 24 hrof isolation. After a further 24 hr, antigen loaded immature monocyteswere induced to mature by culturing with PGE2 (1 μM), IL-1b (10 ng/ml),and TNF-a (10 ng/ml) for 24 hr.

Example 40 Combination Therapy using DHBV Core-TBD and Lamivudine inPekin Ducks

Normal ducklings were infected with DHBV-containing duck serum a dayafter the ducklings were hatched. This is standard practice in the fieldof DHBV research. The presence of persistent viremia was verified usingestablished techniques at week four before the start of theimmunizations. Congenitally DHBV-infected animals at four weeks of agealso were used for the experiments reported herein.

Congenitally DHBV-infected and post-hatch infected ducks were dividedinto three groups. A sample of blood (1.0 mL) was collected forreference of pre-immunization antibody levels and blood samples werecollected every week before the vaccinations. The first experimentalgroup received DHBV Core-TBD chimeric antigen fusion protein 40 μg/doseinjected intramuscularly every other week on the same day until week 22.The second experimental group received DHBV Core protein 19.9 μg/doseinjected intramuscularly every other week on the same day until week 22.The third (control) group received buffer (20 mM Sodium Phosphate pH8.0, 300 mM NaCl) injected intramuscularly every other week on the sameday until week 22. In addition, each group also received 20 mg/kglamivudine injected intramuscularly b.i.d. until week 12, at which pointthe lamivudine dose was increased to 40 mg/kg injected intramuscularlyb.i.d.

No observable local reaction to the injections of the DHBV core-chimericantigen vaccine. No other adverse reaction was noticed. Lamivudine alone(control) decreased serum viremia in both congenitally and post-hatchDHBV infected ducks.

In the control group of ducks, the viremia rebounded at an earlier timepoint compared to the vaccinated group, i.e., ducks receiving DHBVCore-TBD. Thus a trend towards increased viral suppression exists inresponse to vaccination with the chimerica antigen, although a completeelimination of the viremia was not seen in any of the experimentalanimals. A trend towards increased inflammatory response also wasobserved in the group receiving DHBV Core-TBD compared to the controlgroup (lamibviudine alone). Such a trend indicates that the DHBVCore-chimeric antigen induces immune responses in the duck animal model.

In post-hatch DHBV-infected ducks, there was an elevation of serumanti-core antibody levels in core-chimeric antigen treated groupcompared to the control groups. This suggests a humoral response to thevaccination with the chimeric antigen in a chronic virus-infected animalmodel.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled in the artare intended to be within the scope of the following claims.

1-49. (canceled)
 50. A method of producing a chimeric antigencomprising: (a) providing a microorganism or a cell; (b) culturing saidmicroorganism or cell under conditions whereby the chimeric antigen isexpressed.
 51. The method of claim 50, wherein the microorganism or cellis a eukaryotic microorganism or cell.
 52. The method of claim 50,wherein the cell is a yeast cell, a plant cell or an insect cell. 53.The method of claim 52, wherein the chimeric antigen ispost-translationally modified to comprise glycosylation.
 54. The methodof claim 50, wherein the chimeric antigen is post-translationallymodified to comprise a mannose glycosylation.
 55. A method of producinga chimeric antigen comprising: (a) providing a microorganism or a cell,the microorganism or cell comprising a polynucleotide that encodes atarget binding domain bound to a linker molecule; (b) culturing saidmicroorganism or cell under conditions whereby the target bindingdomain-linker molecule is expressed; and (c) contacting the targetbinding domain-linker molecule and an immune response domain underconditions that allow for the binding of the linker to the immuneresponse domain, the binding resulting in a chimeric antigen.
 56. Apolynucleotide encoding a chimeric antigen, said polynucleotidecomprising a first polynucleotide portion encoding an immune responsedomain and a second polynucleotide portion encoding a target bindingdomain, wherein the target binding domain comprises an antibodyfragment.
 57. The polynucleotide of claim 56, wherein the antibodyfragment is a xenotypic antibody fragment.
 58. The polynucleotide ofclaim 56, wherein the polynucleotide comprises a nucleotide sequenceselected from the group consisting of the nucleotide sequences set forthin SEQ ID NOs:39 and 41-51.
 59. The polynucleotide of claim 56, whereinthe polynucleotide encodes a chimeric antigen that is at least 90%identical to an entire amino acid sequence selected from the groupconsisting of the amino acid sequences set forth in SEQ ID NOs:40 and52-62.
 60. The polynucleotide of claim 56, wherein the polynucleotideselectively hybridizes under stringent conditions to a polynucleotidehaving a nucleotide sequence selected from the group consisting ofnucleotide sequences set forth in SEQ ID NOs:39 and 41-51.
 61. A vectorcomprising the polynucleotide of claim
 57. 62. The vector of claim 61,wherein the polynucleotide is operably linked to a transcriptionalregulatory element (TRE).
 63. A microorganism or cell comprising thepolynucleotide of claim 57.